Functional Neuropeptidomics in the Decapod Crustacean: Method

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Functional Neuropeptidomics in the Decapod Crustacean: Method
Functional Neuropeptidomics in the Decapod Crustacean: Method Development and
Application to Behavioral Neuroscience Research
By
Claire Margaret Schmerberg
A dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
(Pharmaceutical Sciences)
at the
UNIVERSITY OF WISCONSIN-MADISON
2012
Date of final oral examination: 12/06/12
The dissertation is approved by the following members of the Final Oral Committee:
Lingjun Li, Professor, Pharmaceutical Sciences and Chemistry
Ronald R. Burnette, Professor and Division Chair, Pharmaceutical Sciences
Ei Terasawa, Professor, Pediatrics
Craig S. Atwood, Associate Professor, Medicine
Arash Bashirullah, Assistant Professor, Pharmaceutical Sciences
©Copyright by Claire M. Schmerberg 2012
All Rights Reserved
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Acknowledgements
I would first like to acknowledge my advisor Lingjun Li, without whom this work would
not be possible. Beyond the initial concepts underlying this work, she has given me the freedom
and support to become an independent scientist. Dr. Li is an outstanding scientist and mentor
who has developed a world-class research program. The creativity and excellence of work in her
lab is without comparison. In the tradition of legendary scientists, Dr. Li allows curiosity to
direct her research program and displays an incredible tenacity to overcome obstacles in
research. This dissertation work directly reflects these qualities: several of the techniques are
challenging to implement to the point that no others have tried them before, and I was able to
investigate fundamental neurobiological concepts in an animal model that is not typically
considered to display such complex behaviors. Dr. Li gave me the freedom to ask the scientific
questions I found interesting, and the support to persevere in challenging situations. She also has
always been supportive in endeavors to improve my professional skills and academic profile. It
has been an honor to work with Dr. Li and I will forever be thankful for this opportunity.
The members of my thesis committee also provided invaluable support and insights
throughout the dissertation preparation process. Their patience and constructive criticism have
raised the academic caliber of this work substantially. I am also very grateful for their support
and regard for my future academic career. I am very thankful to Dr. Ei Terasawa, Dr. Arash
Bashirullah, Dr. Ron Burnette, and Dr. Craig Atwood.
Several other people have contributed directly to this work. This includes my labmates
Zhidan Liang and Nicole Woodards; former labmate Dr. Ruibing Chen; several undergraduates
who have worked with me: Andrew Kozicki, Lauren Putterman, and Kevin Hayes; and Alex
Laztka of the Vander Zanden Lab. I also received helpful input on these projects from Dr.
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Brendan Walker of Washington State University, Kirk J. Grubbs of the Currie Lab, my labmate
Dr. Chenxi Jia, and former labmates Dr. Xiaoyue Jiang, Dr. Robert Cunningham, and Dr. Heidi
Behrens. The UW-Madison Writing Center Mellon-Wisconsin Dissertation Camp was
instrumental in preparing this document and in my professional development. Projects related to
this work but not included in it were aided by a former labmate, Dr. Weifeng Cao, and
undergraduate students: Katherine Zimny and Gajan Muthuvel. The Trout Lake Research Station
from the Department of Limnology, UW-Madison, and Lindsay Sargent of the University of
Notre Dame Environmental Research Center were of great assistance. The Analytical
Instrumentation Center of the School of Pharmacy and the Biotechnology Center provided
equipment. Constructive criticism and support on this work was given by all members of the Li
lab during my tenure here, including several already mentioned and Hui “Vivian” Ye, Dr. Di Ma,
Dr. Robert Sturm, and Zichuan Zhang. Other people in the department who have provided
criticism and support include Gary Girdaukas, Dr. Richard Peterson, and Dr. Steve Oakes.
I am also indebted to the people who first encouraged me to pursue a career in science.
My first research advisors, Dr. So-Hye Cho in the lab of Dr. SonBinh Nguyen and Dr. Bradley
Cooke in the lab of Dr. Catherine Woolley at Northwestern University provided great support to
me in my undergraduate research.
Throughout my graduate career I was supported financially by a variety of mechanisms
for which I am incredibly grateful. These include the School of Pharmacy Kenneth Connors
Wisconsin Distinguished Graduate Fellowship, the Biotechnology Training Program at UWMadison (NIH training grant 5 T32GM08349), the Vilas Conference Presentation Fund, and the
American Chemical Society Women Chemists’ Committee Eli Lilly Travel Award. I have also
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been supported on the following grants to Lingjun Li: NIH grant R01DK071801, and NSF grant
CHE-0967784.
I have been incredibly lucky to have great emotional support, which is mentioned last but
may be the most important factor in completing this dissertation. My boyfriend, Kirk Grubbs,
and my parents, Fred and Nancy Schmerberg, have sustained me in the most challenging times
during my graduate career. At times I was convinced I could not complete this; but Kirk’s and
my parents’ belief that I could kept me from quitting. I also owe a debt of gratitude to other
friends and family members, including my brother Luke and my friends Nicole, Alissa, Charlie,
and Garret. Needed breaks from the mental strain were provided by the Dane County Humane
Society, Eagle Heights Community Gardens, and my pets. I am also grateful to my grandmother,
Betty Robinson Gingras Knapp, who supported me unconditionally and created a family culture
that places a great value on higher education. She was a woman of outstanding courage,
integrity, and compassion. She passed away shortly before I was able to complete this work, but
I know that she would be proud of my achievements.
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Table of Contents
Page
Part I. Introductory Information
Acknowledgements
Table of Contents
Abstract
Chapter 1. Introduction and Overall Summary
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Part II. Background Information
Chapter 2: Neuropeptide Discovery in the Decapod Crustacean
Chapter 3: Methods for Function-Driven Discovery and Functional Assessment of
Neuropeptides
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123
Part III. Development of Tools and Methodology for Functional Neuropeptidomics
Studies
Chapter 4. Affinity-Enhanced Microdialysis for Increased Neuropeptide Recovery
Chapter 5. Mapping the Neuropeptidome of the Species Orconectes rusticus
Chapter 6. Development of Methods for Neuropeptide Quantitation with DataIndependent MS/MS
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207
250
Part IV. Application of Tools and Methodology in Behavioral Experiments
Chapter 7. Application of Microdialysis and Data-Independent MS/MS Quantitation for
Identification of Neuropeptides Involved in Feeding Behavior
Chapter 8. Quantification of Neuropeptides Altered in Acute and Long-Term Ethanol
Exposure
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317
Part V. Conclusions and Supplemental Information
Chapter 9. Conclusions and Future Directions
339
Appendices
A. Protocols for Animal Care, Sample Collection and Preparation, Instrumental Analysis,
and Results Interpretation
B. Crustacean Neuropeptide Database
C. Neuropeptides and Proteins Identified in Orconectes rusticus
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387
419
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Functional Neuropeptidomics in the Decapod Crustacean: Method Development and
Application to Behavioral Neuroscience Research
Claire Margaret Schmerberg
Under the supervision of Professor Lingjun Li
At the University of Wisconsin-Madison
Abstract
Neuropeptides (NPs) represent an important class of signaling molecules whose identities
and functions are not yet fully understood, due mostly to difficulties in studying them using
traditional biochemical methods. These molecules have been implicated in a variety of processes,
including the generation of feeding-related and motivated behaviors, as well as adaptation to
environmental changes. In this work, analytical chemistry methods were developed and applied
to the decapod crustacean (DC), a simple model animal, toward the end of studying timeresolved changes in NPs during behavior.
First, new tools and methods for function-driven neuropeptidomics—an approach to
discover NPs while concurrently gaining information about their function—were developed. The
neuropeptidome of Orconectes rusticus, a DC used in behavioral neuroscience and a potential
model animal for studying environment-neuroendocrine interactions, was described. Affinityenhanced microdialysis was explored to improve sampling of NPs concurrent with behavior. A
new strategy for mass spectrometry data collection that enables simultaneous quantification and
identification (MSE) was developed and applied for the first time to NPs. Proof-of-principle
experiments validated this technique for quantifying NPs that changed after feeding in tissue
extracts of the blue crab, Callinectes sapidus. These tools were then applied to study dynamic
changes in NPs in the Jonah crab, Cancer borealis, under conditions related to feeding and in the
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rock crab, Cancer irroratus, related to motivated behavior. Time-resolved changes in NPs during
feeding in C. borealis were characterized using microdialysis and non-targeted quantitative MSE.
This permitted a close correlation between behavioral and neurochemical changes, allowing
putative assignment of roles for several NPs in food-related reward and ingestive behavior.
Changes in NPs in C. irroratus as a result of acute and repeated exposure to a human drug of
abuse, ethanol, were also characterized. This provides insight into neurochemical changes after
long-term drug exposure, an important factor in the pathology of drug addiction, which is
thought to be a disorder of the natural reward system.
This work not only improves analytical chemistry tools for research into NP
identification and functional determination, but also provides evidence for the potential roles of
several NPs in feeding and motivation-related behaviors. Tools developed in this research will be
useful for behavioral neuroscience and studies of neuroendocrine regulation of environmental
adaptation. This work represents an important step in understanding the basic neurochemistry of
reward and feeding and thus has relevance to basic science as well as human disease.
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Chapter 1: Introduction and Overall Summary
1.1 Introduction
The overall focus of the work presented in this dissertation is the development of cuttingedge analytical chemistry tools for use in neuroscience research, and the application of some of
these tools to study dynamic neuroendocrine processes. It encompasses several areas of research,
both in development and application. Physical experimental tools, a database, and a novel
process for data analysis are developed. For application, feeding behavior and adaptation to
repeated ethanol exposure are studied. The document is divided into five main sections:
introductory information, background information, method development projects, application
projects, and conclusions and supplemental information. In this work, I have striven to improve
upon the promising tools offered by advanced analytical chemistry techniques in order to make
them useful for relevant research into the neuroendocrine correlates of behavior, and have
demonstrated the application of these tools in this field. The introductory information (Table of
Contents, Abstract, and Chapter 1) serves as a general introduction to this dissertation work and
briefly describes each project.
1.2. Background Information
A main focus of this project is the study of neuropeptides (NPs), which are an important
class of neural signaling molecules. Chapter 2 describes these molecules and some methods for
their analysis, and catalogs the known crustacean neuropeptide families with putative functions.
These compounds have many important functions and can act both within the brain and without;
serving as a way for the brain and body to communicate information and coordinate activity.
Although much is known about NPs in mammals, this work focuses on their function in decapod
crustaceans. The simplicity of the crustacean nervous system, both in terms of the number and
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types of compounds present, and in terms of the number and connectivity of the neurons present,
is the main factor in choosing this model organism [1, 2]. The mammalian brain is more
chemically complex due to the presence of additional proteins and peptide fragments of proteins
that occur in vivo and immediately post-mortem [3]. The neuronal substrate is also much simpler
and more extensively studied. Compared to the mammalian brain, which has on the order of 109
neurons, the crustacean nervous system has only thousands, and many in the famous centralpattern generating networks of several ganglia are identifiable and constant in properties and
location from one individual to another [4, 5]. An extensive suite of useful analytical tools based
on mass spectrometry (MS) and tandem MS (MS/MS) techniques has been developed for
analysis of NPs in crustaceans [6], and many NPs have been studied, both for sequence
information and functional information [7]. MS is a highly useful technique for studying NPs
due to its high specificity and ability to rapidly characterize peptide sequences from small
amounts of sample [6]. Chapter 2 enumerates many of the techniques that have been developed
to this end and provides a list of known NPs and their functions.
A new approach for discovery of new NPs, termed function-driven discovery, is
discussed in Chapter 3. This tactic calls for experimental manipulation to elicit changes in NPs
so that functional information is gleaned along with identity information [8]. Function-driven
discovery relies heavily upon quantitative mass spectrometry methods, and a number of these are
described in detail [9]. It also relies upon novel sampling methods, including microdialysis,
which is capable of collecting samples from an animal concurrent with it performing a behavior.
This permits the highest degree of correlation possible between changes in neurochemistry and
changes in behavior short of NP administration [6, 10]. Additional methods for determining NP
function, including those that do not inherently rely upon MS, are also described. This chapter
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describes methods and approaches for using MS to obtain biologically relevant information
along with identification of NPs.
1.3. Development of Tools and Methodology for Functional Neuropeptidomics Studies
In this section, novel tools for NP analysis with functional relevance are developed. A
method to increase the amount of NPs collected with microdialysis, termed affinity-enhanced
microdialysis (AE-MD) is described in Chapter 4. This technique solves some of the challenges
outlined in Ch. 3 in terms of MS quantification sensitivity to enable MD to sample NPs on a
neurobiologically relevant time scale. Microdialysis relies upon diffusion of analytes into a probe
that is surgically implanted into the animal. In the probe, a dialysis membrane is in contact with
the extracellular space, and liquid flowing through the probe carries molecules that have diffused
through that membrane out a length of tubing to where they can be collected and analyzed. AEMD relies upon increasing the rate of diffusion into the probe by adding solid affinity agents that
act as “sinks” for analyte that has diffused into the probe. Thus, the driving force is increased and
additional analyte can be collected [11]. This technique makes quantification of NPs possible
with an experimental setup and condition that was not previously capable of doing so.
Another experimental tool is developed in Chapter 5, a list of the known neuropeptides in
a decapod species of particular interest, the crayfish Orconectes rusticus. This species is used
extensively in behavioral neuroscience and ecology research. It is a model species for studying
aggressive behavior [12-14], motivation-related drug-conditioned place preference behavior [1519], and learning and memory [20-24]. In ecology, it is useful for understanding the properties
that make a species a successful invader [25-29]. Two methods for semi-automated analysis of
MS/MS data to identify NPs are compared, with implications for other neuropeptidomics studies.
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Characterizing the neuropeptidome of this species will enable further research using this useful
model animal.
In Chapter 6, a data analysis approach for obtaining quantitative and identifying
information simultaneously based on a recent advance in MS/MS technology is developed. This
technique uses data-independent acquisition (DIA) MS/MS, which has previously been
demonstrated for quantitation and identification in proteomics studies, where multiple peptides
per protein are obtained [30-34]. This technique is employed with an open-source, vendorneutral software typically used for multiple reaction monitoring (MRM) MS/MS quantification
called Skyline [35]. Skyline is used to generate pseudo-MRM analysis of potentially all analytes
in the sample. The linearity of this quantitation technique is demonstrated and it is applied to a
real sample for comparison to results previously obtained with a different method. This chapter
draws heavily on the concepts of Ch. 3, as DIA MS/MS quantitation is a highly useful tool for
function-driven discovery. In this and other method development chapters, techniques for NP
analysis, ranging from physical tools to data processing methods, are developed for use in
behavioral neuroscience studies.
1.4. Application of Tools and Methodology in Behavioral Experiments
Following development of MS/MS-based tools for NP analysis, these tools are applied to
study two conditions of interest in behavioral neuroscience. In Chapter 7, microdialysis (MD) is
used to sample NPs from the hemolymph of Jonah crabs, Cancer borealis, during feeding
behavior. This process is known to be regulated heavily by NPs in mammals [36], but studying it
in the crustacean may allow for dissection of multiple neural and chemical pathways that overlap
heavily in mammals. This chapter also uses the quantitation technique developed in Ch. 6 for
simultaneous quantification and sequence verification. A total of 28 NPs were quantified, and 5
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were observed to have statistically significant changes throughout the course of feeding. This
chapter demonstrates the applicability of MD and DIA MS/MS quantitation in a behavioral
neuroscience experimental context.
A final application is conducted in Chapter 8, in which NPs in the rock crab, Cancer
irroratus, that change after acute and repeated exposure to ethanol, a common human drug of
abuse, are characterized. It is expected that repeated exposure will lead to adaptations in the
nervous system and its neurochemistry that can be hallmarks of the pathology of substance
dependence or other disorders involving motivation. This chapter takes an untargeted approach
using DIA MS/MS quantitation as developed in Ch. 6, and identifies 22 NPs that may be
potential markers of changes in the neuroendocrine system that are related to long-term drug
exposure. A novel assay for alcohol content in crab hemolymph is also developed.
1.5. Conclusions and Supplemental Information
The final section of this dissertation contains conclusions and potential future directions
that related work could take. It also contains several appendices, including Protocols, a database
of crab NPs developed in Ch. 5 that is applicable for future NP discovery experiments, a
comprehensive list of results from that chapter. These sections are intended to aid future work in
this area, both by listing the procedures developed and improved in this thesis and by tabulating
data that can be used for data processing. The work presented here will provide useful methods
and experimental models for future research into NP identity and function.
1.6. Works Cited
1. Li, L.; Sweedler, J. V. Peptides in the brain: mass spectrometry-based measurement approaches and challenges.
Annu Rev Anal Chem (Palo Alto Calif). 2008, 1, 451-483.
2. Chen, R.; Li, L. Mass spectral imaging and profiling of neuropeptides at the organ and cellular domains. Anal
Bioanal Chem. 2010, 397, 3185-3193.
3. Dowell, J. A.; Heyden, W. V.; Li, L. Rat neuropeptidomics by LC-MS/MS and MALDI-FTMS: Enhanced
dissection and extraction techniques coupled with 2D RP-RP HPLC. J Proteome Res. 2006, 5, 3368-3375.
4. Hooper, S. L.; DiCaprio, R. A. Crustacean Motor Pattern Generator Networks. Neurosignals. 2004, 13, 50-69.
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5. Marder, E.; Bucher, D. Understanding Circuit Dynamics Using the Stomatogastric Nervous System of Lobsters
and Crabs. Annual Review of Physiology. 2007, 69, 291-316.
6. Li, L. J.; Sweedler, J. V. Peptides in the Brain: Mass Spectrometry-Based Measurement Approaches and
Challenges. Annu Rev Anal Chem. 2008, 1, 451-483.
7. Christie, A. E.; Stemmler, E. A.; Dickinson, P. S. Crustacean neuropeptides. Cell. Mol. Life Sci. 2010, 67, 41354169.
8. Schmerberg, C. M.; Li, L. Function-Driven Discovery of Neuropeptides with Mass Spectrometry-Based Tools.
Protein Pept Lett. 2012.
9. Bantscheff, M.; Lemeer, S.; Savitski, M. M.; Kuster, B. Quantitative mass spectrometry in proteomics: critical
review update from 2007 to the present. Anal. Bioanal. Chem. 2012, 404, 939-965.
10. Lee, G. J.; Park, J. H.; Park, H. K. Microdialysis applications in neuroscience. Neurol Res. 2008, 30, 661-668.
11. Duo, J.; Fletcher, H.; Stenken, J. A. Natural and synthetic affinity agents as microdialysis sampling mass
transport enhancers: current progress and future perspectives. Biosens Bioelectron. 2006, 22, 449-457.
12. Tierney, A. J.; Greenlaw, M. A.; Dams-O'Connor, K.; Aig, S. D.; Perna, A. M. Behavioral effects of serotonin
and serotonin agonists in two crayfish species, Procambarus clarkii and Orconectes rusticus. Comp
Biochem Physiol A Mol Integr Physiol. 2004, 139, 495-502.
13. Tierney, A. J.; Kim, T.; Abrams, R. Dopamine in crayfish and other crustaceans: distribution in the central
nervous system and physiological functions. Microsc Res Tech. 2003, 60, 325-335.
14. Panksepp, J. B.; Huber, R. Chronic alterations in serotonin function: dynamic neurochemical properties in
agonistic behavior of the crayfish, Orconectes rusticus. J Neurobiol. 2002, 50, 276-290.
15. Dziopa, L.; Imeh-Nathaniel, A.; Baier, D.; Kiel, M.; Sameera, S.; Brager, A.; Beatriz, V.; Nathaniel, T. I.
Morphine-conditioned cue alters c-Fos protein expression in the brain of crayfish. Brain Research Bulletin.
2011, 85, 385-395.
16. Huber, R. Amines and motivated behaviors: a simpler systems approach to complex behavioral phenomena.
Journal of Comparative Physiology a-Neuroethology Sensory Neural and Behavioral Physiology. 2005,
191, 231-239.
17. Nathaniel, T. I.; Huber, R.; Panksepp, J. Repeated cocaine treatments induce distinct locomotor effects in
Crayfish. Brain Research Bulletin. 2012, 87, 328-333.
18. Nathaniel, T. I.; Panksepp, J.; Huber, R. Drug-seeking behavior in an invertebrate system: Evidence of
morphine-induced reward, extinction and reinstatement in crayfish. Behavioural Brain Research. 2009,
197, 331-338.
19. Panksepp, J. B.; Huber, R. Ethological analyses of crayfish behavior: a new invertebrate system for measuring
the rewarding properties of psychostimulants. Behavioural Brain Research. 2004, 153, 171-180.
20. Acquistapace, P.; Hazlett, B. A.; Gherardi, F. Unsuccessful predation and learning of predator cues by crayfish.
J Crustacean Biol. 2003, 23, 364-370.
21. Hazlett, B. A. Conditioned reinforcement in the crayfish Orconectes rusticus. Behaviour. 2007, 144, 847-859.
22. Tierney, A. J.; Lee, J. Spatial Learning in a T-Maze by the Crayfish Orconectes rusticus. J Comp Psychol. 2011,
125, 31-39.
23. Weisbord, C. D.; Callaghan, D. T.; Pyle, G. G. Associative learning in male rusty crayfish (Orconectes rusticus):
conditioned behavioural response to an egg cue from walleye (Sander vitreus). Can J Zool. 2012, 90, 8592.
24. Tierney, A. J.; Andrews, K. Spatial behavior in male and female crayfish (Orconectes rusticus): learning
strategies and memory duration. Anim Cogn. 2012.
25. Klocker, C. A.; Strayer, D. L. Interactions among an invasive crayfish (Orconectes rusticus), a native crayfish
(Orconectes limosus), and native bivalves (Sphaeriidae and Unionidae). Northeast Nat. 2004, 11, 167-178.
26. Martin, A. L.; Moore, P. A. Field observations of agonism in the crayfish, Orconectes rusticus: shelter use in a
natural environment. Ethology. 2007, 113, 1192-1201.
27. Martin, A. L.; Moore, P. A. The influence of dominance on shelter preference and eviction rates in the crayfish,
Orconectes rusticus. Ethology. 2008, 114, 351-360.
28. Pintor, L. M.; Sih, A. Differences in growth and foraging behavior of native and introduced populations of an
invasive crayfish. Biol Invasions. 2009, 11, 1895-1902.
29. Olden, J. D.; Vander Zanden, M. J.; Johnson, P. T. J. Assessing ecosystem vulnerability to invasive rusty
crayfish (Orconectes rusticus). Ecol Appl. 2011, 21, 2587-2599.
30. Baggerman, G.; Boonen, K.; Verleyen, P.; Loof, A. D.; Schoofs, L. Peptidomic analysis of the larval Drosophila
melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole
time-of-flight mass spectrometry. Journal of Mass Spectrometry. 2005, 40, 250-260.
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31. Finamore, F.; Pieroni, L.; Ronci, M.; Marzano, V.; Mortera, S. L.; Romano, M.; Cortese, C.; Federici, G.;
Urbani, A. Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental
strategies: a comparative study. Blood Transfus. 2010, 8 Suppl 3, s140-148.
32. Levin, Y.; Hradetzky, E.; Bahn, S. Quantification of proteins using data-independent analysis (MSE) in simple
andcomplex samples: a systematic evaluation. Proteomics. 2011, 11, 3273-3287.
33. Pieroni, L.; Finamore, F.; Ronci, M.; Mattoscio, D.; Marzano, V.; Mortera, S. L.; Quattrucci, S.; Federici, G.;
Romano, M.; Urbani, A. Proteomics investigation of human platelets in healthy donors and cystic fibrosis
patients by shotgun nUPLC-MSE and 2DE: a comparative study. Mol Biosyst. 2011, 7, 630-639.
34. Silva, J. C.; Gorenstein, M. V.; Li, G. Z.; Vissers, J. P.; Geromanos, S. J. Absolute quantification of proteins by
LCMSE: a virtue of parallel MS acquisition. Mol Cell Proteomics. 2006, 5, 144-156.
35. MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.;
Liebler, D. C.; MacCoss, M. J. Skyline: an open source document editor for creating and analyzing targeted
proteomics experiments. Bioinformatics. 2010, 26, 966-968.
36. Moran, T. H.; Dailey, M. J. Intestinal feedback signaling and satiety. Physiol Behav. 2011, 105, 77-81.
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Chapter 2: Neuropeptide Discovery in the Decapod Crustacean
Adapted from Schmerberg, C. M., Chen, R., and Li, L. Prog. Neurobiol. 2012 [in preparation].
2.1. Abstract
Invertebrates have played an important role in the discovery and characterization of
neuropeptides (NPs). NPs are a distinct class of neurotransmitters (NTs) that are composed of
short amino acid chains, and they can act both synaptically like classical NTs and systemically in
a manner similar to neurohormones. Discovery and characterization of the identities and
functions of NPs have made great strides in invertebrate model animals, particularly in decapod
crustaceans, due to a number of factors. In this review, advances in techniques for NP
identification and quantitation are discussed, with a focus on mass spectrometry (MS)-based
tools. Assays for determining NP function are also described. Finally, the NP families known
thus far in crustaceans are described in detail. As this article contains both methodology
information and the current state of knowledge with regards to crustacean NPs, it will provide
useful insight to researchers studying NPs in vertebrates as well as in invertebrates.
2.2. Introduction
Neuropeptides (NPs) are a class of neural signaling molecules that are defined by their
composition—short chains of amino acids—and activity—neurochemical signaling or
modulation of signaling. These compounds are important in neural signaling throughout the
animal kingdom and have been implicated in a wide variety of disorders in humans. However,
they have not been studied as intensely as the classical small molecule neurotransmitters (NTs)
for a variety of reasons.
The chemical complexity of NPs poses a great challenge to their study. These molecules
are much larger than NTs, ranging from 500-8,000 Da, and have multiple functional groups
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within their structures. Due to their intermediate size, larger than small molecule
neurotransmitters (NTs) but smaller than proteins, NPs can be more difficult to separate from
other components of the cell. Methods for isolating either small molecules or proteins are quite
mature due to their longer history of study. In addition, the multiple functional groups on the
variety of amino acid side chains of NPs are all potentially reactive. Small molecule NTs
typically contain only a few reactive groups, and many such side chains on proteins are hidden
from the environment by complex tertiary structures. A wider variety of NPs than small
molecule NTs is also possible mathematically, because the molecules can contain 4-20+ amino
acids, each of which has more than 20 possible identities. The size, complexity, and reactivity of
NPs present challenges for their study.
In addition, the genomic information of an organism can be of limited use for NP study
because a number of important processing steps occur between translation and release of a NP at
the synapse or into the circulation (see Fig. 2.1). Preprohormone genes may carry multiple copies
of an NP, or multiple isoforms of NPs with similar sequence motifs (said to be in the same
family). The cleavage of preprohormones into active NPs is also not always predictable.
Although there are several known prohormone processing proteins with known cleavage sites
(such as dibasic AAs), some NPs result from unknown cleavage mechanisms In addition, many
types of posttranslational modifications may affect the activities of NPs, and these modifications
cannot be determined by genetic techniques from gene sequences (reviewed in [1]). Finally, even
in this “post-genomic era”, not all species of interest have sequenced genomes, including all
crustacean species with the exception of the water flea Daphnia pulex [2]. Due to the challenges
of studying NPs, many researchers have turned to simpler model organisms to facilitate their
study through other means. The study of NPs in invertebrates has been instrumental in our
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understanding of these molecules. The simplicity of the blood-brain barrier in many invertebrates
and the presence of well-defined, simple neural circuits have been important factors for using
these species in NP research. The presence of a mature field investigating the effects of
neuromodulators on neural circuits, using one of these well-defined circuits—the stomatogastric
ganglion (STG)—and computational neuroscience methods, is another reason for studing NPs in
crustaceans [3]. Several reviews on crustacean neuropeptides have been published, but most have
focused on a single NP family, or have not discussed the use of cutting-edge analytical chemistry
tools, such as mass spectrometry, in the study of NPs in detail [4, 5]. The following review will
discuss past achievements and recent advances in the discovery and functional analysis of
neuropeptides in invertebrates, focusing on practical aspects, as well as contain a summary of the
known NPs in crustacean species.
2.3. Analysis Methods for Invertebrate Neuropeptide Discovery
Analysis of NPs must take into account the challenges in their analysis mentioned above.
Recent advances in mass spectrometry (MS) have greatly expanded the possible applications of
this technique, and have made it ideal for NP study, particularly in invertebrates. This review
will mention classical biochemistry techniques for NP study, but will focus on MS techniques.
Techniques that have been replaced largely by MS, such as Edman degradation, will not be
discussed in detail.
2.3.1. Immunochemical Techniques
For many years, NPs have been detected with antibodies. Antibodies are used
ubiquitously throughout molecular biology due to their supposed specificity and ease of
visualization. Primary or secondary antibodies can be conjugated with a fluorescent dye,
horseradish peroxidase (HRP), radioactive isotopes, metal (for electron microscopy), or other
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label for visualization or quantification. HRP and fluorescence are most commonly used to
determine the location where the NP is found. Radioimmunoassay (RIA) and enzyme-linked
immuno-sorbent assay (ELISA) are just two of the quantitative techniques that can be conducted.
Although highly specific, high-affinity antibodies can be created, the quality of the
antibody (particularly of polyclonals) will vary from batch to batch, and high specificity is rarely
found in NP antibodies. Often, the portion of the NP sequence that the antibody recognizes is
found on many different NPs, due largely to the presence of common sequence motifs in NPs of
the same family. For instance, many invertebrate FMRFamide-like peptides typically end in the
sequence LRFamide. It has been the authors’ experience that polyclonal antibodies raised against
NPs will have affinity for multiple NPs of the same family, along with other unrelated NPs that
may share some similar sequence motifs, such as an amidated N-terminus. Therefore, it is
impossible to distinguish between the members of a single NP family, all of which may have
distinct functions, by using immunochemical techniques.
2.3.2. Mass Spectrometric Techniques
Mass spectrometry (MS) is a technique that measures the mass to charge ratio (m/z) of
compounds in the gas phase. Taking molecules from solid or liquid preparations and putting
them in the gas phase with charge on them is a major concern in MS, and it is referred to as
ionization. Typically, the process of ionization is harsh to the molecule and induces structural
and chemical changes. In early mass spectrometry of small chemical compounds, a fraction of
the analyte would remain unfragmented upon ionization due to the strength of the chemical
bonds, and thus fragment and precursor masses would be obtained from a single mass spectrum.
The fragment masses could be reassembled into the intact mass to determine the molecule’s
structure. This was not the case, however, with large biomolecules, which are present at lower
12
concentrations and have more labile chemical bonds; only fragment ions were observed with
early ionization techniques, and it was nearly impossible to reassemble these fragments into the
original molecule. With the advent of soft ionization techniques in the late 20th century, it
became possible to conduct mass spectrometric analysis of intact biomolecules with multiple
functional groups and modifications intact. This opened up a new field of biological MS, and
NPs are ideally suited for this analysis. Several strategies for NP analysis by MS-based
techniques are illustrated in Fig. 2.2. Soft ionization techniques include electrospray ionization
(ESI) and matrix-assisted laser desorption ionization (MALDI), among others. In ESI, analytes
in a liquid stream are ionized by spraying the liquid through a small pore with a large electrical
charge applied to it. This causes the molecules to enter the gas phase and to gain electrical
charge(s) in droplets while the liquid solvent evaporates, leaving just charged analytes. MALDI
employs a laser to irradiate a co-crystallized mixture of the analyte and a laser-absorbing
chemical called the matrix. One conceptualization of the mechanism by which MALDI works is
that irradiation of the matrix removes the analytes and matrix molecules from the crystallized
surface in a gas-phase plume, at which time a charge is also imparted to the molecules.
Unlike earlier methods for ionization used in mass spectrometry, these techniques can
impart charge to analytes without fragmenting them, and therefore determination of the m/z of an
intact biomolecule is possible. Adding additional steps of MS analysis permits fragmentation of
the intact molecule and determination of the m/z’s of its fragments, which can then be
reassembled to determine the structure of the original molecule. This is typically done in two
steps as tandem MS (MS/MS or MS2), although more steps of MS analysis can be conducted to
gain further information (n steps of MS, called MSn). For peptides, including NPs, fragmentation
in MS/MS typically occurs along the peptide backbone, and most of the 20 common amino acids
13
(AAs) have unique masses. Thus, the m/z change between peaks can be calculated and correlated
with an AA, and the sequence of the peptide can be determined from the MS/MS spectrum. As
MS identifies the m/z of analytes, it has greater specificity than antibody-based methods, the
pitfalls of which have been discussed above. The sensitivity and quantitative abilities of MSbased techniques have also improved in recent years. Soft ionization MS and MS/MS are the
most specific methods for NP identification. The application of these powerful techniques to NP
study will be discussed in several following sections. A workflow illustrating some of these
techniques in application to studying crustacean NPs is illustrated in Fig. 2.2.
2.3.3. Sampling
Although seemingly simple, the question of how to obtain NP samples from an
invertebrate can be quite complex. The choice of whether to obtain samples from tissues or
physiological fluids (hemolymph in arthropods) has profound implications on the physiological
relevance of the NPs collected and the type of information one can obtain about these NPs.
Samples from tissues typically contain all NPs, including those stored at synaptic terminals,
preprohormones recently translated in the endoplasmic reticulum (ER), intermediate processing
products in vesicles undergoing transport to the axon terminal, and extracellularly released NPs.
In contrast, samples from physiological fluids contain only released NPs. Fig. 2.1 shows the
process by which NPs are synthesized, stored, and released, and demonstrates some of the
possible steps along a NP’s processing pathway. Spatial distribution of NPs is completely lost
when studying samples taken from circulating fluids, unlike tissue samples for which NP
distribution can be mapped with histology or MS. The application of tissue-based and fluidbased techniques, with a focus on preparation for mass spectrometric analysis, will be discussed
in the following section. A schematic showing many of these techniques is found in Fig. 2.2.
14
2.3.3.1. Sampling from tissues
For all tissue-based techniques, the organ of interest is dissected from the animal prior to
being subjected to NP analysis via imaging or survey techniques. Thus, studies in which time or
experimental-related changes are of interest must use different animals for each condition, and
inter-animal variability is a concern. Imaging techniques differ from survey techniques by
showing the spatial distribution of NPs throughout a tissue, whereas survey techniques yield a
single NP profile for the entire tissue.
2.3.3.1.1. Tissue imaging
The development of mass spectrometry imaging (MSI) in recent decades has greatly
advanced the study of neuropeptides. MSI typically relies upon MALDI ionization, which has a
high tolerance for salt and impurities in the sample. This facilitates the detection of bioactive
compounds directly from complex samples and even biological tissues. Using this mass
spectrometric technique, a number of tissue-based neuropeptide discovery methods such as
single cell analysis, direct tissue analysis, and MS imaging (MSI) have been developed. These
techniques combine molecular identification with localization within the animal or even within a
cell. A number of biological tissues have been studied, including single neurons, cell clusters,
ganglia, and single vesicles. Combined with accurate mass measurement or de novo sequencing,
the neuropeptide content in the target specimen can be readily identified without the extensive
sample extraction and purification steps that are required by solution based techniques. A
number of useful reviews of MSI have recently been published [6-12], and this work will serve
as a summary and include perspectives from the authors’ own experience. In the first section,
MSI of large tissues will be discussed.
15
2.3.3.1.1.1. MSI of NPs—Technical Aspects
In MSI, a Cartesian grid is superimposed upon the tissue, and multiple mass spectra are
acquired, at least one for each spot on the tissue. An image can then be generated by processing
this set of MS data with each pixel in the image corresponding to the spectra acquired at that
point. The density of ions at each point can be mapped across the tissue using different colors or
intensities of color to indicate the ion intensity. Following a single acquisition of mass spectra
across the tissue in this manner, ion density maps can be rapidly generated for the entire tissue
for any m/z within the range of MS acquisition, and thus MSI allows for analysis of far more
analytes in a single experiment than histochemical techniques. MSI is particularly attractive in
neuroscience because the location of biologically active molecules can be directly related to their
functions, and MSI is also helpful for investigation of expression changes that occur as a result
of experimental perturbation or animal development. The spatial distribution of target molecules
obtained by MSI can be lost when tissues are homogenized for some other types of MS analysis
techniques. A recent paper employing MSI is of particular interest [13].
Two types of technologies are typically used in MSI: matrix-assisted laser
desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS). The choice of
technique is dependent on the spatial resolution desired, the chemical properties of analytes of
interest, and the instrumentation available. For optimal spatial resolution, SIMS imaging has
submicron precision. However, its method of ionization relies upon a high energy (5-25 keV)
primary ion beam (Ca+, In+, Cs+, Aun+ etc.) to bombard the surface of the sample and produce
ions for MS. Not only does this require specialized equipment, the high energy of the ion beam
can break down the target molecules, particularly large biological compounds, into smaller
fragments. Fragmentation is less common in MALDI MS due to ionization by laser irradiation,
16
and thus larger intact molecules can be detected, with the tradeoff of reduced spatial resolution
(~50 µm). For imaging techniques, depending on the thickness of the specimen, tissue can be
placed directly on the sample plate, or frozen and sliced by a cryostat into 5 to 15 µm sections
prior to transferring it to the plate. This is the full extent of sample preparation for SIMS. In
MALDI-MS, a matrix must be applied to the sample prior to instrumental analysis. This is a
crucial aspect of sample preparation, because deposition of MALDI matrix in must be
homogeneous to avoid significant migration of analytes on the surface of the tissue or the
formation of “hot spots” due to more or less matrix present in a single location. Some methods of
matrix application that limit migration and uneven coating include robotic micro-spotters or
modified printers creating dense arrays of droplets [14], sublimation of matrix onto tissue, and
airbrushes for matrix application. The sample is then ready for data acquisition. Data is acquired
in a grid pattern with a predetermined step size (e.g. 100µm), depending on the sample size and
required resolution. Most often, specialized software is used to acquire and process data. Notable
software for MSI includes BioMap (developed by Novartis and available free of charge at
www.maldi-msi.org) and vendor-specific software. The processing software can generate heat
map ion images of target m/z values by plotting the intensity of a single compound in each mass
spectrum against the x, y coordinates of each pixel.
Over the last decade, numerous developments have been achieved to improve the quality
of MALDI imaging results, both in sample preparation methodologies and instrumentation. For
sample preparation, a number of approaches have been tried, including altering the tissue itself or
the matrix, to improve sensitivity and/or spatial resolution. Ethanol has been commonly used for
tissue fixation [15]. By rinsing with serial ethanol-containing solutions, physiological salts and
impurities can be removed from the tissue to improve spectral quality. Additionally, ethanol can
17
fix the proteins in the tissue sample in place to eliminate redistribution of these proteins. A final
benefit of ethanol fixation is greater stability over time. However, based on the observations in
our laboratory, rinsing with ethanol may cause migration of hydrophilic NPs [13]. Glycerol
stabilization has also been employed to preserve cell morphology and prevent migration of NPs
[16]. In addition to treatment of tissue, sample thickness has also been investigated as a factor in
the sensitivity of MSI for biomolecules. Sugiura et al. systematically investigated serial mouse
brain sections with a range of thicknesses, and concluded that thinner tissue sections (<10 µm)
were optimal for MSI [17]. Matrix-related improvements to MSI have also been explored. For
example, depositing gold on the tissue surface increased the signal intensities in the peptide mass
range [18].
Resolution is another important aspect that is usually limited by matrix crystal size and
the dimension of the ionizing laser beam. A plethora of methods and devices have been used to
reduce matrix crystal size, and only a few examples will be discussed here. One matrix
application method reported by Sugiura and colleagues combined spraying and dispensing
droplets. This led to more homogeneous crystals and increased mass spectral peak intensity [19].
Sold ionic matricies have been created from the mixture of a conventional MALDI matrix with
an organic base (e.g. aniline). These matricies improved spectral quality, crystal homogeneity,
and resistance to laser irradiation (i.e. they were not ablated rapidly) [20]. A large amount of
effort has gone into improving sensitivity and spatial resolution for MSI focusing on the sample
and matrix.
Improvements to the spatial resolution of MALDI-MSI data by alteration of laser properties
are also of note. The diameter of a laser beam is usually 50 or 100 µm for commercial
instruments equipped with Nd:YAG or N2 lasers. The oversampling technique can be used to
18
improve spatial resolution to a size smaller than the laser diameter by altering the way in which
images are acquired. First, the matrix in one location is completely ablated. The sample plate is
then moved at a step size that is smaller than the laser diameter, and spectra are acquired again
until matrix is ablated, and so on. Thus the spectra acquired are from an area with size equal to
the small step size the plate was moved, which is much smaller than the laser size [21]. This
technique is valuable for studying small neural structures. New laser developments are also
capable of improving spatial resolution. Compared to classical Nd:YAG or N2 laser, the
Smartbeam laser developed by Bruker Daltonics has an adjustable focus size ranging from 10 to
80µm, which allows a smaller step size to be used during experiments. A combination of
improvements in sample preparation, matrix deposition, and lasers have improved the sensitivity
and spatial resolution of MSI over the past several years, but these remain as major challenges to
consider in MSI experiments.
2.3.3.1.1.2. MSI of NPs—Recent Advances and Comparison to Traditional Techniques
MALDI-IMS has been used to map a variety of compounds in tissue, including
cholesterol and phospholipids [22], proteins [23], and pharmaceuticals [24, 25]. More recently,
the use of IMS to map the distribution of neuropeptides, particularly in mammalian neural
tissues, has gained attention. In one study, SIMS and MALDI imaging were both employed to
map the distribution of NPs, lipids, and small molecules in rat spinal cord. This study determined
that several NPs—including substance P and somatostatin-14—were colocalized [26]. MSI has
been used to profile the NPs in several invertebrate species, notably in Aplysia. Its millimetersized ganglia have been profiled, and the peptide contents of cell bodies and neuronal processes
(neurites) in single isolated Aplysia neurons have been compared [16, 27]. Crustacean species
have also been subjected to MSI. Multiple neuropeptide families in the brain and pericardial
19
organ of the crab Cancer borealis have been mapped in detail [13]. Three-dimensional MALDI
imaging of the brain of C. borealis was also recently presented by Chen and coworkers. The
simple scheme employed was able to generate a thorough description of neuropeptide
localization in the crab brain. In the image obtained, a great degree of correlation is seen between
NPs and the identified structures of the crab brain [28]. MSI has had recent success in imaging
the distribution of NPs, particularly in invertebrate tissues.
Traditionally the spatial localization and relative amounts of bioactive peptides are
visualized with immunostaining or in situ hybridization. Immunostaining uses fluorescent,
enzyme-linked, or radiolabeled antibody to probe the location of target proteins or peptides in the
tissue. It has the advantage that the precise peptide sequence is not required, and its high
resolution enables visualization of subcellular distribution. However, one main drawback exists
to this method—the specificity of antibodies, which has disadvantages described previously.
Frequently, multiple neuropeptides (typically from the same NP family) react with a single
antibody raised against a single NP. Thus, extensive rounds of antibody development may be
required to obtain an antibody that is specific for a single NP, and in many cases it may be
impossible to create such an antibody. This type of experiment is also unsuitable for discovering
novel NPs because the compound must be identified before an antibody can be made for it. For
in situ hybridization, a nucleotide probe binds to the target gene transcript and is visualized,
frequently through fluorescent tagging of the probe. Thus, this technique also requires prior
knowledge of the target nucleotide sequence, which is again unsuitable for novel NP discovery
and difficult to carry out in organisms without known genomes. MSI is more flexible than
immunostaining or in situ hybridization because the peptides of interest do not need to be
preselected. The sample preparation of MSI is also relatively simple without the need for
20
extensive washing and fixation. Furthermore, numerous target molecules with diverse chemical
structures can be analyzed simultaneously, and their colocalization can be visualized simply to
achieve valuable information regarding interactions between different neuropeptides.
2.3.3.1.2. Tissue Survey
Survey techniques include homogenization followed by extraction, single-cell analysis of
large molluscan neurons, and direct tissue profiling of small organs in the decapod crustacean
nervous system. These are illustrated in Fig. 2.2. Of these three, homogenization followed by
extraction is the most common, and can be used in conjunction with both MS and
immunochemical detection. One or more of the desired organ or organs are collected and the
tissues are homogenized. NPs are then extracted from the homogenate with a solvent that
selectively solubilizes NPs over lipids and proteins, such as acidified methanol. This sample is
then subjected to a number of clean-up steps (delipidation, desalting, ultracentrifugation) and
analytical separations (LC, electrophoresis) to reduce the sample complexity prior to NP
analysis, which is conducted by MS or immunochemistry. Fig. 2.3 illustrates some of the sample
preparation techniques that can be used on liquid samples.
2.3.3.1.3. Direct Tissue, Single-Cell, and Single Vesicle Analysis
Direct tissue profiling and single neuron analysis—techniques based on MALDI-MS—
are useful in obtaining a “snapshot” of the NP content of a small organ or single neuron. In these
methods, the tissue or single neuron to be characterized is carefully dissected, then washed,
placed on a MALDI plate, and coated with the desired matrix. The strength of this method lies in
the ability to determine which NPs are present in a small organ or large moluscan neuron with
minimal sample preparation steps. This increases the sensitivity and speed of analysis over
extraction techniques.
21
2.3.3.1.3.1. Detailed Information on Sample Preparation for Direct Tissue and Single Cell
Analysis
As the first step of these studies, tissue dissection is crucial to the quality of the acquired
mass spectra. Molluscan neurons (50-500 µm) have been demonstrated to be suitable for singlecell analysis. For smaller cells, micromanipulation techniques using micro glass capillary or
small diameter glass micropipettes are required to isolate the cells and transfer them to the
sample plate [29]. When the cells are too small or difficult to dissect, a small piece of tissue or
neuronal cluster can be analyzed directly using similar methods to MSI, in which the small tissue
is subjected directly to laser irradiation for ionization.
Sample preparation also plays a pivotal role in this type of analysis. The physiological
salt concentrations associated with neurons from marine specimens or the saline solution used
for dissection greatly interferes with MALDI analysis. These concentrations are about four times
higher in marine crustaceans compared to mammals. Several simple rinsing procedures have
been developed to enable direct assay for peptides with minimal sample handling. Garden et al.
employed on-target water washing of Aplysia neurons prior to MALDI in order to reduce the
high levels of salts found in this marine mollusk [30]. Extracellular salts and physiological saline
can also be removed in a stepwise fashion by spot-to-spot cell transfers to a fresh matrix drop on
the sample plate. Li and colleagues employed concentrated 2, 5-dihydroxybenzoic acid (DHB)
matrix solution in 4:1 acetone/water mixture for a method of this fashion, termed on-target
micro-extraction [31]. An alternative simplified preparation method involves replacing the
physiological saline with the DHB matrix solution during dissection, so that the salt removal
occurs simultaneously with neuronal isolation. For direct tissue analysis, rinsing samples first in
acidified methanol followed by dilute DHB (10mg/ml) aqueous solution has been shown to
22
efficiently remove physiological saline and extract the neuropeptides from the cell for sensitive
MS detection [32]. Sample preparation for small tissue analysis mostly involves methods to
remove salts present in the animal under normal conditions, as these physiological
concentrations of salt are more than sufficient to inhibit ionization in MALDI.
Once the sample is dissected and prepared, the following procedure is relatively simple.
For matrix deposition, the dried-droplet method can usually be used with success. One of many
widely used MALDI matrices can be chosen, such as 2, 5-dihydroxybenzoic acid (DHB) or
alpha-cyano-4-hydroxycinnamic acid (CHCA) for peptide analysis, and sinipinic acid (SA) for
protein analysis. In the authors’ experience, DHB is preferred for tissue-based analysis for
several reasons. These include stabilization of cell membranes for improved microdissection,
reducing proteolytic enzyme activity, and replacement of salt ions present in the tissue [30].
Storage in a DHB solution is also able to preserve the tissue for up to several years without
obvious degradation of the NP content [33]. MALDI matricies can be used during or after tissue
dissection to improve spectral quality for small tissue analysis with a number of advantages.
However, sensitivity is a crucial concern for analyzing direct tissue and single cell
samples, due to the miniscule sample amount. Sample dilution induced by matrix spreading can
reduce signal intensity and cause poor spectral quality. It is reported that deposition of matrix in
nanoliter volumes over a period of 10 s can be used to eliminate spreading [34]. Wang et al.
modified the MALDI plate with a parafilm coating to improve the sensitivity of detection by
concentrating the samples on target via the solvent-repellent effect of the parafilm surface [35].
In addition, several novel small-scale sample preparation techniques have been developed
recently to ease the complicated dissection procedure for single cell analysis while increasing the
detection sensitivity. For instance, neuropeptide content in neurons and ganglia can be easily
23
extracted by incubating them in an aqueous solution of DHB. The resulting signaling moleculecontaining solution can be directly examined by MALDI MS [33]. A DHB solution-based
capillary electrophoresis separation method was also developed to couple with MALDI analysis
of liquid incubated with small neuronal tissues or single cells [36]. This separation prior to
analysis will improve the dynamic range of detection and increase the number of analytes
detected by reducing sample complexity. In an additional innovation to assay large numbers of
individual cells for comprehensive profiling of tissue slices or organs, a massively parallel
preparation of single-cell-sized samples was developed by placing a thin tissue section on an
array of glass beads attached to parafilm. When the parafilm is stretched, the tissue is divided
into thousands of small pieces, which can be analyzed separately [37]. This mechanical
separation allows greater spatial resolution in imaging of the tissue than would previously have
been possible. Although most sample preparation techniques for small tissue analysis can induce
analyte dilution, specialized matrix deposition techniques and separation, both chemical and
mechanical, can increase sensitivity.
2.3.3.1.3.2. Recent Developments in Direct Tissue and Single Cell Analysis
The study of individual identified cells is extremely valuable for neuropeptide discovery,
since fewer analytes are present in a single cell compared to tissue extracts. Furthermore, it is
also important to investigate chemically heterogeneous nervous systems in which cells specialize
in the synthesis of functionally and/or chemically distinct signaling molecules. Thus knowledge
of neuropeptide complements on a cellular basis is essential for understanding their bioactivities.
The first single-cell MALDI-MS study was performed using identified neurons from the
Lymnaea stagnalis brain [38]. The mollusk is a good choice for improving sample preparation
techniques for single cell analysis, since the relatively large size of their neurons and greater
24
simplicity of their nervous system makes dissection and related sample handling less challenging
than in other species. Since then, various freshwater and marine mollusks have been studied,
including Helix aspersa, Aplysia californica, Aplysia vaccaria, Phyllaphlysia taylori, and
additional work in Lymnaea stagnalis [39-42]. In these studies, a large range of NPs have been
identified, such as insulin-related peptides, FMRFamide-related peptides and small cardioactive
peptides. For further review, see recent papers by Li and Hummon [43, 44]. Single-cell analysis
using molluscan neurons has made significant advances in our ability both to analyze small,
complex samples and in our understanding of their neuronal NP content.
With improvements in sample preparation methods and increases in performance of MS
instruments, many more organisms have been studied in recent years. Ma and coworkers
reported the first single cell study of individual insect neurons by analyzing pheromone
biosynthesis activating neuropeptide (PBAN) in subesophageal ganglion neurons (diameter
around 20 µm) from the corn earworm moth Helicoverpa zea [45]. Recently Neupert et al.
performed dissection and MS analysis of identified neurons and neurosecretory cells from the
American cockroach Periplaneta americana. In these studies, a large number of FMRFamiderelated peptides (FaRPs) [46] and allatotropin-related peptides (ATRP) [47] were detected.
Neupert and coworkers also reported the first detection of tick periviscerokinin from single cells
in Ixodes ricinus and Boophilus microplus [48]. On an even smaller scale, single vesicles from
the atrial gland of Aplysia californica have been analyzed by Rubakhin and coworkers. A wide
range of bioactive peptides within individual vesicles (1-2 µm) were identified, including
products from several genes colocalized within the same vesicle [49], which runs counter to the
canonical model of how NPs are loaded into vesicles. Other species have been successfully
25
subjected to single-cell MALDI analysis for NP content, including the nematode Ascaris suum
[50] and the rat Rattus norwegicus [51].
In addition to studies using single cells or organelles, various reports have been published
analyzing neuron clusters or endocrine nerve tissues. For these samples, direct tissue analysis is
widely used, especially for smaller cells that are difficult to isolate. This method is a simple and
fast way to achieve a snapshot of the neuropeptide content in a target organ. For example,
multiple putative tackykinin-related peptides in Drosophila were identified by directly analyzing
the anterior-ventral brain using MALDI MS [52]. Direct tissue analysis (analysis of discrete
points, as opposed to IMS of all possible points) is also extremely useful to acquire a complete
mapping of neuropeptide distribution throughout the whole animal. For example, Yew et al.
detected numerous neuropeptide families in several major neurohemal structures in nematodes,
and a body-region-specific distribution of these neuropeptides was observed [53]. Larger tissues
can be analyzed using direct tissue profiling to rapidly obtain profiles of the NP content of an
entiretissue or of discrete spots within the tissue.
This method is also extensively used for neuropeptide discovery in crustaceans. The
neuroendocrine tissues, pericardial organ (PO) and sinus gland (SG), from several different
crustacean species were analyzed by Li et al. using MALDI-TOF, and found to contain a large
number of putative neuropeptides [54, 55]. In more recent studies, neuroendocrine organs
isolated from the crab Cancer borealis were analyzed by a MALDI Fourier transform mass
spectrometry (FTMS) instrument using an in-cell accumulation technique. This technique
improves the detection sensitivity by accumulating ions from many subsequent laser shots within
the cell prior to analysis, which is particularly helpful for analytes from a complex tissue sample
[32]. This same MALDI-FTMS platform was also used for analysis of large peptides, such as
26
crustacean hyperglycemic hormones (CHH), in studies that incorporated an in-cell cleanup step
to remove matrix adducts, which facilitated detection of these large molecules [56]. Direct tissue
analysis has been used, along with a wide variety of other sample preparation techniques, to
detect and characterize hundreds of neuropeptides in the crustacean [57-59]. Several putative
tachykinin-related peptides have also recently been characterized in midgut epithelial tissues and
several neural organs in multiple Cancer species, and the gut-brain distribution of these peptides
appears to be conserved [60]. Direct tissue MALDI analysis has been an instrumental technique
for crustacean NP discovery and localization.
2.3.3.1.4. Comparison of Tissue-Based Techniques
All three types of tissue-based sampling techniques for mass spectrometry analysis have
unique advantages. Imaging is the only method to obtain a detailed map of the distribution of one
or more NPs throughout an organ. Direct tissue and single neuron analyses yield a quick
snapshot of the NP content of the sample, and can be conducted with less material, which in turn
leads to increased detection sensitivity. NP distribution information obtained from imaging,
direct tissue, and single neuron analysis can be integrated with functional information already
known about specific ganglia, sub-structures of these ganglia, or identified neurons. For instance,
an NP found solely in the olfactory bulb of the crab brain, either by MSI or direct tissue analysis,
may have a functional role in the chemical senses and/or how they affect other neuronal
processes. Thus, direct tissue, imaging, and single cell analysis techniques allow for preliminary
assignment of NP function based on the known roles of certain subsets of cells to which the NPs
are localized. In contrast, homogenization followed by extraction reduces the functional
information content of the sample by combining NPs from many cells that may have individual
functions into a single sample—all spatial distribution information is lost. Solid-phase ionization
27
techniques are the only option for intact tissue analyses, however, and these techniques often
suffer from run-to-run irreproducibility and poorer quality MS/MS spectra due to singly charged
ions.
Homogenization and extraction of neural tissues has advantages over intact tissue-based
techniques in the variety of treatments that the samples can undergo to improve the quality and
sensitivity of analysis. Chief among these are liquid-phase reactions and analytical separations,
illustrated in Fig. 2.3. For higher-mass NPs, an enzymatic digestion step is valuable to generate
fragments with masses within the range where modern mass spectrometers have high sensitivity
and resolution. Enzymatic reactions can also be carried out to characterize certain posttranslational modifications. Other reactions that are most efficiently conducted on samples in the
liquid phase include labeling reactions to improve MS/MS fragmentation, identify functional
groups, and incorporate isotopes for more reliable MS and MS/MS quantitation. Although
reactions can be conducted on the surface of solid samples, this is less efficient and can lead to
diffusion of NPs away from their original location in the tissue. Enzymatic reactions conducted
on tissue samples for histochemistry are less common.
A variety of techniques to reduce sample complexity, such as immunoprecipitation (IP),
delipidation, desalting, ultracentrifugation, ultrafiltration, and dialysis are also most amenable to
liquid samples (illustrated in Fig. 2.3). These techniques are capable of enriching neuropeptides
by antibody-antigen interactions, hydrophobicity, and molecular weight. Additional liquid-based
separation techniques, such as LC (from preparative to UPLC scale) and electrophoresis
(capillary or gel), are compatible with homogenized and extracted NPs, but not with intact
tissues. Removing salts, lipids, and high molecular weight compounds allows for better
ionization of samples in MS. IP, LC, and electrophoresis separate analytes to decrease the
28
complexity and increase the sensitivity of analysis. Extracted samples from multiple animals can
also be pooled to increase sensitivity and allow detection of low-abundance analytes.
When using MS, liquid-based ionization methods can be used to introduce extracted NPs
into the mass spectrometer. Chief among these is electrospray ionization (ESI), which has the
advantage of adding multiple charges to a single NP. This increases the quality of fragment ions
observed in a subsequent MS/MS spectrum and facilitates de novo sequencing. ESI also is more
reproducible from run-to-run than solid-phase ionization techniques, which improves
quantitative analysis.
2.3.3.2. Sampling from Biological Fluids in Invertebrates
Liquid-based sampling relies upon withdrawing a biological fluid from the experimental
animal and analyzing this substance for the presence of NPs. Fig. 2.3 illustrates some methods of
obtaining liquid samples from invertebrates. For invertebrates, this usually takes the form of
hemolymph sampling. This substance is analogous to blood in invertebrates, and bathes all
organs due to the open circulatory system of invertebrates. Extremely small samples of
hemolymph can be used, such as the hemolymph from a single fruit fly [61]. All of the above
solution-based sample cleanup, labeling, and separations techniques can be conducted on this
fluid, depending on the volume obtained, which can then be analyzed by MS or immunochemical
techniques. However, hemolymph withdrawal can be challenging and can expose the animal to a
great deal of stress. Many small invertebrates can only be used for a single hemolymph sample
due to the limited volume. Aquatic invertebrates must be removed from the water for sampling,
which exposes them to a great deal of stress and potentially hypoxia.
Dilution of NPs in hemolymph and the complexity of hemolymph itself also decrease the
sensitivity of detection. NPs released into this fluid may be present at a concentration that is
29
orders of magnitude less than the concentration at the site of release or action. For instance,
many NPs are postulated to act in the micro- to nanomolar range at their receptors. However,
diffusion from the micro-environment of immediately adjacent to the cell into the hemolymph as
a whole dilutes these compounds into a much larger total volume. For this reason, detection of
NPs in hemolymph with high-specificity tools such as mass spectrometry is challenging. As an
example, hundreds of NPs have been detected in tissue extracts from Cancer borealis [35, 56,
59, 62-66], but studies where sampling has been conducted from the hemolymph have only
identified tens of these NPs [67-69] (summarized in Table 2.1).
The hemolymph also contains many high-abundance proteins and protein fragments in
vivo, which can obscure the detection of low-abundance NPs. In the same hemolymph studies
mentioned above [67-69], only 11 NPs could be detected in hemolymph collected directly from
the crab via needle, and fragments of two high-abundance proteins (cryptocyanin and actin)
dominated the mass spectra obtained. With the sample collection technique of microdialysis
(MD), the number of NPs detected increased to 35 with fragments of only one high-abundance
protein collected. This technique allows sampling from the hemolymph by utilizing a MD probe,
which is implanted into the animal with the tip of the probe in contact with the hemolymph. At
the tip of this probe is a semipermeable dialysis membrane, through which molecules below a
certain molecular weight (the molecular weight cutoff or MWCO) can diffuse into the probe.
Liquid is constantly pumped through the probe at a low flow rate, and this liquid carries the
molecules that have diffused through the probe tip through the outlet tubing. The liquid that
flows out can then be collected for analysis. In this situation, the main advantage of MD is the
exclusion of high molecular weight compounds from the sample, which leads to a less complex
sample with fewer proteins that can degrade and interfere with the detection of low-abundance
30
NPs. However, high-abundance protein fragments are still detected in MD samples, indicating
that these fragments must be present in the hemolymph in vivo.
2.3.3.3. Comparison of tissue-based to liquid-based techniques
2.3.3.3.1. Advantages of liquid-based techniques
The two advantages of liquid-based sampling are the ability to sample only NPs released
into the extracellular space, and the potential to sample multiple times from the same animal (for
larger invertebrates). Sampling only NPs in the extracellular environment yields more
biologically relevant information about these compounds, including the final bioactive form
released by the cell for signaling. Tissue-based sampling, on the other hand, utilizes
homogenization, a laser beam, or detergents to disrupt cell membranes, and thus the NPs
collected represent not only those released in response to a particular stimulus, but those that are
being translated, processed, or stored inside the cell. These compounds’ sequences may not
represent the final bioactive form, but some prehormone along the post-translational processing
pathway. Tissue-based sampling techniques sample inactive prepro- and pro-peptides, stored
peptides, and released peptides, whereas liquid-based techniques only sample those NPs released
into the extracellular space.
In addition to revealing the active form of the NPs, sampling from the extracellular
environment also provides more insight into the regulation of NP release by a certain
physiological process. Tissue-based techniques can only detect net changes in the NP content of
the cell, which are due to the sum of NP release and changes in the amount of pre-prohormone
translation. It is thus difficult to tell from tissue sampling by what mechanism the NP is regulated
in response to a physiological change. In contrast, the only factors that influence NP
concentration in the extracellular fluid are release from cells and degradation over time, which is
31
usually a zero-order process not tied into the animal’s response to stimuli. By sampling from the
hemolymph, we are better able to determine if a NP is released by cells in response to a certain
change.
Not only does fluid sampling improve the quality of data obtained about NP release, but
it permits multiple samples to be taken from a single animal. This decreases problems with interanimal variability in the study of dynamic physiological processes. Neuropeptides are known to
be involved in both rapid synaptic communication and long-term physiological modulation, and
thus the temporal course of their release is of great importance when attempting to determine
their function. A great deal of inter-animal variability is observed in baseline NP concentrations,
both in tissue extracts and liquid samples. Comparing the net change in NP concentration in a
single animal provides more relevant information about NP function. Baseline concentrations of
NPs can change several-fold between individual invertebrates. The change in NP concentration
in response to a stimulus is often 1-2 fold, and thus lies within the inter-animal variability if
different animals are used for each time point around the stimulus. Sampling from the same
animal multiple times makes it possible to see NP concentration changes that would otherwise be
lost in the inter-animal variation at each time point. Liquid-based sampling has the distinct
advantage of allowing for repeated sampling from the same animal to observe changes in NPs
that are obscured due to high baseline variability in NP concentration between individuals. For
these reasons, liquid-based sampling is advantageous when studying rapid changes in NP
concentration associated with function.
2.3.3.3.2. Advantages of tissue-based techniques
Tissue-based techniques have the advantage, however, when distribution of an NP is of
interest. Due to the open circulatory system of most invertebrates, NPs that are present there are
32
not localized to any one region of the body. Any association with a particular organ or subsection of an organ is lost when sampling from the hemolymph. With tissue-based techniques,
the localization of NPs throughout the nervous system can be determined. This can be important
in well-defined nervous systems, such as ganglia with a known function (i.e. the pericardial
organ as a neurosecretory organ), subsections of ganglia associated by connectivity with a certain
function (i.e. the antennal lobe in the crustacean brain), or in a ganglion for which the
connectivity of each neuron in a circuit is known (i.e. the stomatogastric ganglion of the
crustacean). In addition, the local neurotransmitter roles of some NPs will not be reflected in
hemolymph concentrations of these compounds, as they will be present only at synapses in the
tissue. Any NP released at a synapse is not likely to diffuse into the hemolymph in amounts that
can be detected with precision. When the localization of an NP throughout the body of an
invertebrate is important, tissue-based techniques are preferred over fluid-based sampling.
2.4. Bioinformatics Tools for Discovery of Invertebrate Neuropeptides
2.4.1. Peptide Mass Matching for NP Discovery
Once spectra have been acquired, they must be interpreted to determine the identity of the
compounds present. The simplest way to identify neuropeptides from MS spectra is by searching
experimental masses against a known neuropeptide database. This can be achieved by comparing
with a list of known neuropeptide masses via manual comparison or by automated search. Public
neuropeptide
information
databases
are
currently of
great
use,
such
as
SwePep
(http://www.swepep.org/) [70] and the Endogenous Regulatory OligoPeptides (EROP) database
(http:// erop.inbi.ras.ru./). SwePep is designed for identification of mouse endogenous peptides
by their mass based on Uniprot annotation, although it is no longer updated regularly. EROP
database contains the known bioactive peptides from information of the literature. This type of
33
analysis is analagous to peptide mass fingerprinting of a digested protein, a commonly used
approach in proteomics.
As mentioned previously, neuropeptide prohormones are processed into multiple gene
products by the actions of a number of enzymes. Bioinformatics tools have been developed to aid
in predicting the final peptide forms from a prohormone; these tools use binary logistic
regression models trained on known neuropeptides to identify cleaved basic sites among the
many possible cleavage sites that exist in a prohormone [71, 72]. Thus, one can make wellinformed decisions on the expected neuropeptides from a novel prohormone. These prediction
tools are accessible via http://neuroproteomics.scs.uiuc.edu/neuropred.html [73], which also
contains lists of prohormones and their associated peptides for a variety of common neuronal
models.
As described above, there are two common methods of performing MS analysis of brain
tissues — direct profiling via MALDI and ESI-MS/MS of extracts. The direct profiling of a cell
or a tissue has an advantage in that many of the peptides from a specific prohormone are detected
at the same time. When multiple peptides are detected using accurate mass, even if MS/MS
cannot be obtained, the ability to confidently assign the peaks is enhanced. Single cell MS is an
extreme example where often a significant fraction of a prohormone sequence can be covered
with accurate mass MALDI MS measurements.
2.4.2. Strategies for MS/MS Sequencing of NPs
For extracts or larger brain regions, one gains the ability to work with more complex
samples but loses the advantage of prohormone coverage. In such cases, the ability to sequence
novel neuropeptides can be critical. Neuropeptide sequencing requires an instrument capable of
producing fragment ions (via MS/MS). Depending on instrument availability and the species of
34
interest, a number of different data analysis strategies can be employed for MS/MS neuropeptide
identification.
First of all, if the species of interest has a sequenced genome, a standard protein database
search can be performed using proteomic-based search engines, such as Mascot (Matrix Science,
http://www.matrixscience.com/) or SEQUEST (Thermo Corp., http://www.thermo.com/) [74].
Although these search engines were designed for proteomics applications, they are easily
adapted to neuropeptidomics. The proteins in the database are virtually digested into peptides.
Then the fragmentation spectrum is compared with these virtual peptides. The software can also
take into account PTMs and selects the most likely peptide using a scoring function. The
problem with this method is the lack of a virtual enzyme that has exactly the same cleavage
pattern as the protein convertases that cleave neuropeptide precursors. Considering all possible
cleavage sites and PTMs is thus required, although it increases the false positive rate, prolongs
the search time, and reduces the identification confidence.
If the species of interest does not have a sequenced genome, de novo sequencing of the
neuropeptide is usually required. In contrast to database searching, de novo peptide sequencing
uses no a priori information, and instead relies upon direct ‘reading’ of the amino acid sequence
from the MS/MS spectrum. With a great deal of experience, de novo sequencing can be done
manually. However, this is extremely labor intensive and time consuming. Fortunately, several
software packages exist which perform de novo sequencing directly on MS/MS data, including
LuteFisk (http://www.hairyfatguy.com/lutefisk/), PepSeq (packaged with Waters’ MassLynx
software, http://www.waters.com/), Mascot Distiller (Matrix Science), PEAKS (Bioinformatics
Solutions,
http://www.bioinformaticssolutions.com/),
(http://proteomics.ucsd.edu/Software/PepNovo.html), and others [75].
PepNovo
A number of these
35
programs, such as PEAKS and Mascot Distiller, include automated or batch de novo processing
to enhance sequencing speed and flexibility.
Another way to identify neuropeptides employing a database is by sequence tag
searching. Finding the complete sequence from fragmentation spectra is only possible when
spectra of high quality have been obtained. In contrast to Mascot and SEQUEST, which base
identifications on parent masses and MS/MS fragmentation patterns, sequence tag searches are
performed using small contiguous strings of identified amino acids (sequence tags) compared
against sequences in the database [76]. Even if the exact protein is not present in the database,
there is a high likelihood that homologues of the protein can be found. Some sequence tagging
programs can handle homologue mutations or possible sequencing errors. Since this technique
does not rely on parent mass identification, it is a powerful technique for identifying NPs
containing PTMs. For example, MS-Seq (Protein Prospector, http://prospector.ucsf.edu/) and
Mascot offer sequence tag searching for single tag queries. Both of these interfaces are flexible
and powerful but can be time consuming if analyzing a large volume of data.
2.4.3. Verification of MS/MS Sequencing for NPs
Basic
Local
Alignment
Search
Tool
(BLAST)
homology
search
(http://www.ncbi.nlm.nih.gov/BLAST/) can be also performed to confirm the identification of
neuropeptides from de novo sequencing. A BLAST search compares a partial sequence against
the database of a closely related species. While a homology search rarely yields a complete
sequence, it can provide useful information about evolutionary origins and potential function of
the partially sequenced SP. More recently, the use of pattern finding software, such as SPIDER
(Bioinformatics Solutions) and MEME (http://meme.sdsc.edu/meme/) has improved partial
sequence homology searches [77]. These programs are optimized for MS/MS derived data and
36
are more tolerant to sequencing errors than BLAST searches. We certainly expect that
bioinformatics tools tailored to the unique features of NPs will continue to expand.
Every sequencing method can introduce faults. It is argued that the combination of
multiple search methods can be used to reduce the false positives and increase the coverage and
confidence for peptide identification, since different search engines have different criteria of
interpretation, and if two or more independent methods produce the same result, then it is more
likely the result is accurate. The combination of Mascot and SEQUEST has been demonstrated
to improve the sensitivity and accuracy of protein identification in shotgun proteomics
experiments [78]. Indeed, more and more software programs are beginning to incorporate this
consensus functionality. For example, the most recent PEAKS software package incorporates
five different search engines, including Mascot, SEQUEST, OMSSA, X!Tandem, and PEAKS
Protein Identification. The software can combine the results to generate a unique report, which
provides better coverage and accuracy for peptide and protein identification.
2.4.4. Genome Mining for NP Discovery in Crustaceans
Recently, a number of cDNA libraries have been constructed for crustacean species with
the goal of allowing for gene-based studies of their physiology. For many of these libraries,
expressed sequence tags (ESTs) have been generated and submitted to publicly accessible
databases [79, 80], creating a rich resource from which to mine potential proteins of interest. In
silico analysis methods have been established to discover novel neuropeptides from these
databases. These methods use known prepro-hormones or known peptide sequences as queries to
search for transcripts putatively encoding neuropeptide precursor proteins using the BLAST
program tblastn (search of translated nucleotide database using a protein query). The transcripts
thus obtained are subjected to processing via several on-line protein programs to predict the
37
mature forms of the peptides contained within them, which are then verified via homology to
known peptide isoforms. This strategy has been used successfully for peptide discovery in
insects [81-83], and has been used to study neuropeptides in crustaceans. For example, in a
recent study, 12 unannotated ESTs encoding putative neuropeptide prepro-hormones were
identified from six different species [84]. Predicted processing of the encoded precursor proteins
revealed over 60 putative neuropeptides. The use of in silico analysis can also help to resolve the
ambiguity of MS/MS data interpretation for de novo sequencing. For example, it permits
assignment of leucine and isoleucine in amino acid sequences, which cannot be conducted with
MS/MS due to the fact that these two residues are isobaric, and it also can confirm previously
discovered neuropeptides. With the continued addition of new ESTs to publicly accessible
databases, undoubtedly other neuropeptide precursors will be included among them, and as
additional in silico searches are conducted, more neuropeptides will be discovered, providing a
foundation for future molecular, mass spectral and physiological investigations.
Recently, the first crustacean genome was published for the Cladoceran Daphnia pulex
[2]. A number of papers have used genome mining in combination with biochemical and mass
spectrometric techniques to characterize NPs present in D. pulex. Neuropeptides from multiple
families have been identified from genomics information [85-87]. In some cases,
immunohistochemistry has been used to verify the presence of a certain NP in this animal [87].
Further studies have verified NPs predicted from genomic analyses by MS/MS [86]. Finally, the
a putative red pigment concentrating hormone (RPCH) identified from the D. pulex genome has
been synthesized and used in a pigment dispersion assay in another species to determine how its
activity varies from forms of RPCH previously identified in other crustaceans [88]. Nearly every
family of crustacean neuropeptide has been identified from genome mining in D. pulex.
38
However, some remain elusive. Further refinement of genome searching algorithms may provide
additional information about those NPs not yet identified from the genomic information. In
addition, the sequencing of additional crustacean genomes will be a great boon to NP discovery.
Although Daphnia is an important crustacean genus in environmental assessment, it is of limited
use in understanding neuropeptide function due to its small size and limited set of behaviors.
Sequencing the genomes of decapod crustaceans, where more is already known about NP
sequences, would be of great utility.
2.6. Known Crustacean Neuropeptides
A large number of NPs have been discovered and characterized in many crustacean and
other invertebrate species. The following section will discuss current knowledge about the
identities of these NPs, their distribution throughout the body, physiological functions of these
NPs, and molecular mechanisms by which they act. Due to the large number of NPs identified
and the wide variety of decapod crustacean species studied, this section cannot contain all
current knowledge, but an attempt has been made to include the most influential and/or recent
findings. NPs are often classified into families of similar compounds based on shared amino acid
sequence motifs, and each family will be discussed separately.
2.6.1. RFamide-Related Peptides
The first RFamide peptide—and the first identified invertebrate neuropeptide,
FMRFamide, was characterized from the mollusk Macrocallista nimbosa in the late 1970s by
Price and Greenberg using multiple chromatographic separations followed by Edman
degradation and various biochemical techniques [89]. It was initially recognized for its
cardioexcitatory/myoactive activity, determined in functional assays where either natural or
synthetic FMRFamide was applied to the isolated ventricle of Mercenaria mercenaria or on the
39
radular protractor muscle of Busycon contrarium [89]. In the following years, a large diversity of
FMRFamide related peptides (FaRPs) possessing arginine (R) and amidated phenylalanine (F) as
a C-terminal motif have been found throughout the invertebrate animal kingdom, observed in
Nematoda, Annelida and Arthropoda, among others. RFamide peptides show a remarkable array
of physiological functions, including as the well-known cardiovascular activity, along with
modulation of sensory organs, motility, reproduction, and feeding. The diversity of possible
functions is likely a consequence of the large diversity in their amino acid sequences.
Since the first identification of FMRFamide in M. nimbosa, around 30 isoforms of
FMRFamide related peptides (FaRPs) have been discovered in several major classes of mollusks
using both traditional biochemical methods and mass spectrometric methods. A recent review
includes most of the progress in FaRP characterization in the phylum Mollusca [90]. FaRPs have
also been extensively studied in nematodes, especially Caenorhabditis elegans. The completion
of the C. elegans genome sequencing project was achieved in 1998, and the complexity of NP
signaling in this simple nematode is illustrated in studies mining this genome. Overall, 23 genes
encoding FaRPs (as known as flp genes) were initially found in C. elegans [91, 92]; and a total of
59 distinct putative FaRPs can be predicted from processing of these 23 genes, as multiple
mature FaRPs forms can be produced from each of the propeptide precursors. By systematic
BLAST searching of the nematode EST database, conducted by McVeigh et al., the total number
of identified nematode flp genes has increased to 31. The study by McVeigh and colleagues
predicted an additional 25 novel distinct FaRPs from these findings [93]. A recent twodimensional LC–MS/MS study analyzed whole body extract from C. elegans of multiple
developmental stages [94]. This work identified 25 putative FaRPs and characterized several
common posttranslational modifications, including amidation, pyroglutamic acid C-termini, and
40
methionine oxidation [94]. Other animal models from the phylum Annelida have also been
analysed for FaRP content, such as the earthworm [95] and leech [96].
2.6.1.1. FMRFamide-Related Peptides in Crustaceans
The FaRPs are also present in crustaceans as one of the families with the most diverse
amino acid
sequences.
In the first
study of these peptides
in
the crustacean,
immunocytochemistry of eyestalk tissue from the prawn Palaemon serratus revealed that this
tissue contains a compound that reacts with a polyclonal anti-FMRFamide antibody [97].
Through the following two decades, using similar techniques, FaRPs were detected in numerous
crustacean species, including Cancer borealis, Panulirus interruptus, Homarus americanus, and
Procambarus clarkii. FMRFamide-like immunoreactivity has been observed in all major
neuronal organs in the crustacean, including the central nervous system (CNS), the STNS, and
the neurosecretory organs: pericardial organ (PO) and sinus gland (SG).
However, accurate characterization of FaRPs in the crustacean was hindered due to the
difficulty of performing accurate and high throughput sequence analysis. At the beginning of the
21st century, only a few FaRP isoforms had been characterized using the microsequence analysis
technique (Edman degradation) [98-100]. In recent years, with the development of MS
techniques, a large number of FaRPs have been discovered, revealing the high complexity of this
peptide family in crustaceans, similar to the observed complexity of the flp genes in C. elegans.
For example, in a recent neuropeptidomic study of C. borealis, Ma and coworkers identified 41
FaRPs along with more than 100 neuropeptides from other families [59]. Based on several largescale mass spectrometric neuropeptide characterization studies, the number of FaRPs identified
in the crustacean has greatly increased in recent years. [57, 59, 101-103]. These peptides can
usually be divided into three different groups based on their C terminal motif, including –
41
FLRFamide, -YLRFamide, and –RLRFamide, with distinct localizations and physiological
functions.
FaRPs havealso been extensively studied in insects, including the cockroach Periplaneta
americana [104], the grey flesh fly Neobellieria bullata [105], andthe fruit fly Drosophila
melanogaster [106]. The division of insect FaRPs into subfamilies follows different rules than in
the crustacean. FaRPs in insects are divided into four subfamilies, including -FMRFamide
containing peptides, myosuppressins (XDVXHXFLRFamide), sulfakinins (-DYGHMRFamide,
Y represents a sulfated tyrosine) and short neuropeptide F (SNPF, -RLRFamide). Some of those
peptides are remarkably conserved between insects and crustaceans. For example, sulfakinins
[107] and SNPFs have both been isolated and characterized in several crustacean species in
addition to the insect species in which they were originally described. Furthermore, the –
FLRFamide containing peptides in crustaceans show great structural similarity to insect
myosuppressins. It is important to note, however, that peptides containing the exact sequence FMRFamide have not been observed in crustaceans as yet, despite the large number of FaRP
family members described in these species.
2.6.1.2. Functions of FMRFamide-Related Peptides
As described above, FaRPs are present in many different organisms with a large diversity
of amino acid sequences. These peptides also exert a large array of physiological functions, from
modulating synaptic transmission within neural circuits to acting as hormones in the circulating
fluid to affect behaviors. Many studies have shown that FaRPs are vital in several major aspects
of the animal’s life, such as reproduction and feeding, and these functions are often conserved
between different organisms at different evolutionary levels. Several articles have reviewed their
functions specific to certain phyla or organisms, such as mollusks [90], insects [108], nematodes
42
[109]and crustaceans [110]. A general and cross-species discussion will be given in this paper
regarding the most recent progresses.
The first isolation and characterization of FMRFamide arose from an investigation into
its cardioactivity in mollusks, and thus members of this family are known primarily as
cardioactive peptides. The regulatory functions of FaRPs on cardiac outputs have been well
documented in many different organisms. They can also modulate the contraction of many other
types of smooth muscle tissue tissue in diverse species. For instance, FaRPs have been shown to
modulate the alimentary tract and hindgut ganglia of B. amphitrite [111], pharyngeal motility in
H. medicinalis [112], and crop movements in D. melanogaster [113].
FaRPs are also known to positively and negatively regulate the reproductive axis in
vertebrates by modulating the gonadotropin-releasing hormone (GnRH) neurons. Different
FaRPs stimulate (kisspeptin) and inhibit (gonadotropin-inhibiting hormone (GnIH)) this axis
[114]. A great deal of evidence has also demonstrated that FaRPs play a regulatory role in
reproduction in invertebrates as well. In Octopus vulgaris, the short peptide FMRFamide was
observed to modulate the effect of dopamine on photoreceptors in animals adapted to light or
dark conditions. In both light- and dark- adapted animals, dopamine + FMRFamide caused the
retinae to quickly adapt to light exposure, which has the overall effect of inhibiting sexual
maturation. [115]. Several FaRPs, including FMRFa, FLRFa, FIRFa, and an extended FLRFa,
have also been found to play a role in peptidergic control of egg-laying in the cephalopod Sepia
offcinalis by regulating oocyte transport through the oviduct [116]. Recently, it was
demonstrated that mutations in the G protein coupled receptors for certain FaRPs in C. elegans
cause inhibition of egg-laying [117], again perhaps due to action on muscles in the oviduct, or
more direct functions on the reproductive axis.
43
Another important role identified for FaRPs in several species is to regulate feeding. In
crustaceans, –FLRFamides are able to modulate the rhythmic contractions of several parts in the
digestive system. In 1990, a cholecystokinin (CCK)-like hormone (vertebrate homolog of
sulfakinin) was found to activate the gastric mill rhythm in the lobster, levels of a peptide that
reacted with anti-CCK increased in the hemolymph following feeding, and a CCK antagonist
inhibited feeding-induced gastric mill rhythms [118]. Furthermore, Mercier and colleagues
detected increased FaRP immunoreactivity in the hemolymph of crayfish 1 hour following
feeding [110]. Additional support for the role of FaRPs in feeding regulation comes from
evidence that FaRPs modulate the rhythmic outputs of several food intake and digestion-related
central pattern generators in Aplysia [119]. Studies in decapod crustaceans and the mollusk
Aplysia suggest a role for FaRPs in feeding.
A number of studies in insects also support a role for FaRPs in feeding behaviors. Hill
and Orchard reportedthat under different diet and feeding states the expression level of FaRPs in
cockroach gut tissue changed [120]. In addition, different FaRP family peptides occasionally
affect the visceral muscles in ways that are opposite but are necessary for overall food
processing. For instance, insect sulfakinins stimulate the foregut muscles, and myosuppressins
inhibit spontaneous contractions of the foregut. However, both sulfakinins and myosuppressins
inhibited feeding in vivo [121-123]. The role of FMRFamide-related peptides in feeding is
complex and may be due to their more well-known myoactive properties. It is clear, however,
that FaRPs play some role in feeding in several invertebrate species, including insects,
crustaceans, and Aplysia. Other putative functions for FaRPs include regulation of reproduction,
which is more commonly seen in vertebrates but has also been observed in two cephalopod
44
species and one nematode. The canonical function of FaRPs is muscle stimulation, which has
been demonstrated on the heart and several portions of the digestive tract.
2.6.1.3. Molecular Mechanism of FMRFamide-Related Peptide Action
It is well known that the activity of most FaRPs is mediated mostly through G-protein
coupled receptors (GPCRs). However, studies of neurons in mollusks revealed that FMRFamides
cause a fast depolarizing response by activating a sodium channel on the cell membrane. The
observation of FMRFamide-induced inward currents when the GPCR-blocking agent guanosine
was present indicates that a FMRFamide-gated ion channel exists. First cloned from the snail
Helix, this FMRFamide-gated sodium channel (FaNaCh) has also been observed in several other
species reviewed in [124, 125]). So far, FaNaCh is the only known ionotropic receptor that is
regulated directly by peptides in invertebrates. Interestingly, a vertebrate cationic channel gated
by the FaRP kisspeptin has also been found, and it is a member of the transient receptor potential
channel type C (TRPC) family [126-128]. Direct control of ion conductance by NPs is rare, and
it may be significant that vertebrate and invetertebrate FaRPs both act via this mechanism. The
physiological implications of FaNaCh are still elusive and further study will be necessary to
determine the significance of this nonclassical mechanism of NP action. A crustacean GPCR for
FaRPs has not yet been identified, and thus its molecular action through this receptor is also
currently unknown.
2.6.2. Allatostatins
2.6.2.1. Types of Allatostatins Found in Crustaceans
The allatostatin (AST) peptides are named for their first identified function. In insects,
ASTs inhibit juvenile hormone synthesis in the corpora allata [129]. Many ASTs have since been
identified in various invertebrates [101, 130, 131], and they are now identified as allatostatins by
45
sequence homology to the first described ASTs. Three main groups exist, based on sequence tags
of peptides that modulate the corpora allata in different insect species. A-type allatostatins
possess the common C-terminal pentapeptide motif Y/FXFGL-NH2 and were first identified in
the cockroach [129, 132]. B-type ASTspossess the C-terminal sequence W(X)6Wamide, with X
being variable amino acids and were first found in the cricket [133]. Finally, C-type ASTs
possess a nonamidated, conserved C-terminus –PISCF and were first isolated from Manduca
sexta and Lepidoptera [134]. Several reviews have presented the structures of these peptides,
their allatostatic and other functions, and the organisms in which they have been found,
especially focusing on insect species [135-138].
It was long believed that only A-type ASTs were present in crustaceans. The first
crustacean A-type AST was found in the crab Carcinus maenas [139]. Recently, more than 100
crustacean A-type allatostatins have been reported using both biochemical methods and mass
spectrometric techniques in multiple crustacean species [32, 57, 59, 101, 102, 130, 140, 141],
showing a large degree of sequence diversity. Recently, a gene encoding this peptide family was
cloned in the freshwater prawn, Macrobrachium rosenbergii. This gene encodes a massive
polypeptide precursorcontaining 701 amino acid residues. This prepro-AST is capable of
producing 35 potential mature A-type AST peptides [142]. In keeping with the A-type AST
family sequence tag, these peptides contain YAFGL as the primary C terminus. Using state-ofthe-art mass spectrometry, the concept of only A-type ASTs being present in crustaceans was
refuted, as a B-type AST was identified in Cancer productus POs [101]. Following this
discovery, additional B-type AST isoforms were identified in numerous crustacean species [59,
143]. The existence of non-A-type ASTs in crustaceans was further supported by recent
discoveries of two C-type AST isoforms [64, 144]. In contrast to the diversity of A- and B-type
46
AST isoforms, there are usually only one or two isoforms of C-type ASTs found in each
organism, similar to insects. The variation in the primary sequences of ASTs may result in
different physiological activities. Immunohistochemistry studies show that ASTs are widely
distributed in both the CNS and stomatogastric nervous systems (STNS) of various crustacean
species [130, 145, 146]. Additional research has mapped ASTs throughout several
neuroendocrine structures, including the pericardial organ, sinus gland and perineural sheath of
the thoracic ganglia [140, 146, 147]. Finally, ASTs have also been detected at relatively low
concentrations in the hemolymph [67, 68, 148]. These observations suggest ASTs can function
via both neural and humoral pathways, with both neurotransmitter and neurohormonal roles.
2.6.2.2. Allatostatin Function in Crustaceans
The signature role of ASTs in insects is regulation of development and reproduction by
inhibiting juvenile hormone (JH) synthesis in the corpora allata (CA). In crustaceans, the
mandibular organs (MO), located at the bottom of the mandibular tendon, are homologous to CA.
MO can produce the sesquiterpenoids methyl farnesoate (MF) and its immediate precursor,
farnesoic acid (FA). MF is a precursor to insect JHs that may serve the same role in crustaceans
as JH does in insects. It has been reported that treatment with Dippu-ASTs on isolated MO from
adult crayfish, Procambarus clarkii, increased MF synthesis by MOs that were not actively
producing large levels of MF [148]. This study goes on to show that ASTs do not affect FA
levels, and thus the authors suggest that ASTs act on the enzyme that converts FA to MF.
Observations in this study are contradictory to the canonical role of ASTs in inhibition of the CA
in insects. However, non-classical roles for ASTs in insects are similar to the observed increase
in MO activity seen in this study. As yet, there is no evidence that suggests ASTs act as
inhibitors as well as stimulators of MF synthesis in crustaceans, however very few studies have
47
been performed and only A-type AST has been tested. Further investigation is required to fully
understand the physiological functions of ASTs on regulation of sesquiterpenoid biosynthesis in
crustacean and arthropods in general.
The currently accepted nomenclature of allatostatin is based on the initial discovery of
one function of these groups of peptides in insects–the inhibition of juvenile hormone
biosynthesis by the corpora allata. However, ASTs also have numerous other functions,
including activity modulation in the central pattern generator (CPG) networks of the crab
nervous system. In an isolated STNS preparation, Diplotera allatostatins were able to reduce the
pyloric rhythm frequency. This was dose-dependent and the amount of reduction was related to
the initial frequency [145]. Although the C-terminal sequences of the three subtypes of ASTs
vary greatly, they have been shown to act in similar ways on the STNS [64, 143]. These Cterminal motifs may also be the most functionally relevant parts of the ASTs, as the C-terminal
pentapeptide of A-type ASTshas been shown to produce activity at its receptor [149]. The action
of ASTs on electrically excitable cells has also been demonstrated using A-type ASTs on the
neuromuscular junctions of several crustacean species. The application of these compounds led
to reductions in the amplitude of signals transmitted and the movements thus elicited [150, 151].
A-AST has also been shown to induce action in the cardiac ganglion (CG) CPG, responsible for
contraction of the heart. In isolated CGs from C. borealis, it strongly inhibits the motor
output[152]. This finding demonstrates yet another CPG in which A-ASTs reduce activity,
leading to the hypothesis that A-ASTs may be important chemical messengers for reduced
activity in the heart, in other neural circuits, and in excitable cells in general. A contradictory
result was obtained from application of C-type AST to H. americanus heart preparations. The
amplitude of the heartbeat was instead increased in a state-dependent manner. This may indicate
48
that A-and C-typeASTs have opposite effects on heart function. A-type ASTs have also been
shown to alter sensory neuron activity [153]. Finally, an inhibitory endocrine role for ASTs on
the rhythmic hindgut contractions in many arthropod species has been described [154-156].
ASTs have also been detected in the gut tissue in crustaceans [142, 157], however their
physiological activities have not been tested. In general, it appears that AST-type peptides inhibit
excitable cells throughout the bodies of invertebrates. It should be noted, however, that most
studies investigate A-type ASTs, and there is some evidence that different AST subfamilies may
have opposing functions.
2.6.2.3 Molecular Mechanisms of Allatostatin Action
Allatostatin receptors are GPCRs similar in structure to those of the mammalian
somatostatin/galanin/opioid receptor family [158]. A unique receptor family has been identified
for each of the A-, B- and C-type AST subfamilies in Drosophila [159]. The first identified AST
receptors were found in Drosophila, which carries two genes that encode GPCRs for A-type
AST peptides. Both of these A-AST receptors are members of the rhodopsin-like GPCR family,
whose closest vertebrate homolog is the galanin receptor family [158, 160]. B-type AST
receptors and vertebrate bombesin receptors appear to be most related [161]. Due to the presence
of a disulfide bridge at the C-terminus of C-type ASTs, they are thought to be homologous to
somatostatin. Additional support for this homology comes from the similarity between their
receptors [162, 163]. Due to the specificity of different AST subtypes for different receptor
families, functional similarities in AST actions are more likely to occur in downstream signaling
molecules. Drosophila AST activates G protein-coupled inwardly rectifying potassium (GIRK)
channels [158]. This decreases cellular excitability by hyperpolarizing neurons and reducing
their input resistance [163, 164]. This reduced excitability is a possible explanation for the
49
inhibitory actions of A-, B- and C-type ASTs on rhythmic activity in the STG. Because GPCRs
are commonly desensitized following prolonged activation, typically by phosphorylation or
internalization [165], long-term exposure to a single type of AST might eventually lead to a loss
of efficacy. Release of another form of AST could continue to propagate the signal, as it would
act upon different receptors, but have similar downstream targets. This may be an explanation for
the diversity of AST isoforms present and their similar release profiles. AST peptides appear to
have distinct receptor types for each subfamily, but in general lead to reduced excitability. This
divergence of receptors but convergence of effector proteins along with the diversity of ASTs
present but similarity in their release and functional relevance may constitute a method to
provide continuous signaling even in the presence of receptor desensitization.
2.6.3. Tachykinins and related peptides
The tachykinin NP family was first identified in vertebrates. They can function as
brain/gut peptides that affect both central and peripheral tissues. Some well-known mammalian
tachykinins include substance P and neurokinins, which possess the C-terminal motif of FXGLMa. The first invertebrate tachykinin Lom-TK-I was characterized from brain of Locusta
migratoria. By the early 2000’s, around forty invertebrate tachykinins (Inv-TKs) and tachykininrelated pepides (TKRPs) had been characterized from a wide range of invertebrates, including
insects, crustaceans, worms and mollusks. Some earlier studies were reviewed in articles from
Severini et al. and Satake et al. [166, 167]. More recent studies, particularly those in crustaceans,
will be the focus of this review.
2.6.3.1. Tachykinins and Related Peptides in Invertebrates
Most TKRPs contain the primary sequence motif –FXGYRa. Using mass spectrometric
techniques, several novel TKRPs have been recently identified. In insects, TKRP is a large
50
family with numerous isoforms. A number of studies have identified these peptides in various
Heteropteran species. In one such study, six different TKRPs were de novo sequenced from the
antennal lobes of seven different species, five of the Pentatomoidea superfamily (shield and
stinkbugs) and two others. The five Pentatomoidea species studied—Nezara viridula, Euschistus
servus, Acrosternum hilare, Banasa dimiata, and Pentatoma rufipes—share the same TKRPs,
whereas the other two more distantly related species—Oncopeltus fasciatus and Pyrrhocoris
apterus—have distinct TKRPs [168]. Studies in two cockroach species identified two TKRP
precursors that encode all of the nine previously reported LemTRPs in addition to four novel
ones [169]. A study in the honey bee, Apis melifera, was able to definitively identify 4 different
TKRPs by using a combination of genomic database mining and mass spectrometric analysis
[170]. Typically, insect species have many different TRP isoforms.
Crustacean tachykinin related peptides (CabTRPs) were first identified in the CNS of the
crab C. borealis. In these seminal studies, two forms were identified: APSGFLGMRa (CabTRP
1a) and SGFLGMRa (CabTRP 1b) [171]. CabTRP 1b is the less potent of these two in a
cockroach hindgut contraction bioassay. The 1b isoform does not alter the pyloric rhythm of the
STG, whereas CabTRP 1a does. CabTRP 1a is released from the STG under normal
physiological conditions in Cancer borealis [172]. It was also found in the anterior commissural
organ in Cancer productus, using both immunohistochemical and mass spectrometric methods
[173]. Using direct tissue analysis, several CabTRPs have been detected from midgut tissue in
three closely related crustacean species, Cancer magister Dana, Cancer productus Randall, and
Cancer borealis Stimpson. In the same study, the midgut epithelial endocrine cells were found to
be a rich source of CabTRPs [174]. Recently a novel TKRP, TPSGFLGMRa, was identified
inthe midgut and STNS of Cancer irroratus, C. borealis, C. magister, and C. productus.
51
Notably, it was not detected in Panulirus interruptus and Procambarus clarkii. The application
of this peptide to the C. borealis STG elicited an identical pyloric motor pattern as CabTRP 1a
[60]. This novel peptide was later detected in the American lobster Homarus americanus [175].
CabTRPs have also been detected in the hemolymph of C. borealis, which hints at a possible
hormonal role for this peptide family [67, 174]. In a recent large-scale MS neuropeptidomics
investigation of C. borealis, TPSGFLGMRa and its methionine oxidation-modified form were
detected in the CNS and STNS [59]. Methionine oxidation is thought to be a modification that
occurs during sample handling, not in vivo. Overall, only three CabTRPs and their
posttranslationally modified peptides have been discovered in different crustacean species so far
in contrast to the large numbers of isoforms observed in insect species, but they are found
extensively in the CNS, STNS, midgut, and hemolymph; and at least two of these isoforms have
identified functions.
2.6.3.2. Functions of Tachykinin and Related Peptides
The mammalian tachykinin peptides, such as substance P and neuropeptide K and many
others, are known to have a diverse range of biological functions, including but not limited to
transmission of pain signals, various facets of reproduction, inflammation, immune system
function, and food intake [176, 177]. In invertebrates, the TKRPs are found in both nervous
tissue and digestive tissue, and many have been shown to modulate gut function, as described in
several reviews focusing on insects [108, 178]. The stimulatory effects of TKRPs were first
demonstrated in the hindgut of the cockroach, and TKRPs have since been shown to produce
visceral muscle contraction in other insects. It has also been reported that Lom-TKs can cause
contraction of the oviduct in L. migratoria [108]. Electrophysiological experiments on the crab
STNS have found that application of CabTRP Ia elicits the pyloric rhythm or increases several
52
aspects of this rhythm in preparations where the rhythm is already active [171]. Therefore,
tachykinin-related peptides may play an important role in feeding and/or digestion in the
crustacean. A cardiac effect of TRPs is also suggested by evidence that CabTRP 1a increases the
burst frequency and duty cycle in the cardiac ganglion [152].
Immunohistochemistry studies have documented the expression of TKRPs in the
olfactory lobes of both insects and crustaceans in detail. Winther et al.reported that TKRPs affect
odor perception and locomotor activity in Drosophila [179]. In this work, it was demonstrated
that RNAi knockdown of the TKRP prehormone gene altered responses towards specific
odorants and different odorant concentrations. Hyperactivity in locomotion assays was also
observed in these knockdown flies. Quantitative MS analysis of A. melifera brains following
foraging behavior demonstrated dramatic changes in the levels of TKRP expression. This may
indicate a role for TKRPs in food collection in social insects, although the importance of
olfaction in foraging and previous implication of TKRPs in olfaction may be an alternative
explanation for these results [180]. A number of insect studies suggest that TKRPs stimulate
food consumption when released from midgut into the hemolymph. This hormonal role for insect
TKs has been suggested by the presence of TK immunoreactivity in the endocrine organs of the
midgut in Leucophaea maderae [108]. In the cockroach, decreases in LemTRP-1 levels in the
midgut were associated with higher levels of food intake. Additionally, starvation led to lower
levels of LemTRP-1 in midgut but increased levels in hemolymph [108]. Furthermore, in studies
where LemTRP-1 was injected into live cockroaches, orexigenic effectswere observed [181].
These findings suggest that midgut endocrine cells release LemTRP-1 into the hemolymph to
increase feeding.
53
In addition to central and peripheral functions in adult animals, TKRPs are also involved
in neural circuit development in both vertebrates and invertebrates [166, 182, 183]. As an animal
matures, the output of its neural networks changes due to a number of factors. These include
changes in the underlying networks, the modulatory inputs to these networks, and/or the effects
of neuromodulators on the networks. In the lobster H. americanus, TKRPs are not observed in
the STG of embryos and only begin to be observed in later stages, during which adult-like motor
patterns also start to occur. TKRPs are expressed much later than other neuropeptide families
[184]. A possible explanation for the reduced complexity of STNS output in embryonic lobsters
is that not all neuromodulators, in particular TKRPs, are present at early developmental stages.
Application of CabTRP 1a to isolated embryonic and adult STNS leads to changes in the STG
motor patterns in both stages; however, this effect is different between the two stages. It is
therefore likely that the receptor for CabTRP 1a is present in the embryonic STNS, despite the
immaturity of the network in total [185]. Production of the NP itself is restricted to
developmental stages in which the neuronal connectivity has matured. It is therefore possible that
CabTRP 1a production is a signal of a mature neural network.
2.6.3.3. The Tachykinin Receptor
The receptors for TKRPs belong to the GPCR superfamily, with the phospholipase C
(PLC) -inositol triphosphate (InsP3)-calcium signal cascade as the intracellular mediator of the
signal. So far five TKRP receptors (TKRPRs) have been identified in invertebrates: including
DTKR (Drosophila melanogaster) [186], NKD (Drosophila melanogaster) [187, 188], STKR
(Stomoxys calcitrans) [189], UTKR (Urechis unitinctus) [190], and oct-TKRPR (Octopus
vulgaris) [191]. Studies of the structure and distribution of TKRPRs are essential to understand
the physiological functions of these neuropeptides. In O. vulgaris the mRNA of oct-TKRPR was
54
detected in both the central nervous system and peripheral tissues, such as several smooth
muscles including stomach, rectum, ovary, etc. This pattern is similar to the distribution of
TKRPs in other invertebrate animals, and further implicates these peptides in a wide spectrum of
physiological processes such as midgut contraction, feeding and reproduction [191]. Further
research in crustaceans into the tachykinin receptor and its intracellular signaling pathways may
provide additional insight into the importance of tachykinins in crustaceans.
2.6.4. Orcokinins
The orcokinins were originally purified from the nervous system of the crayfish
Orconectes limosus [192], and it is this species for which they are named. These NPs have been
shown to modulate hindgut contraction with high potency, and have been detected by
immunochemistry in various neural structures, including the STG, in several crustacean species
[193]. The cDNA of two preproorcokinin genes has been isolated from the brain tissue of the red
swamp crayfish, Procambarus clarkii. These genes encode 6 unique orcokinins, whose
sequences have been verified via mass spectral techniques [194]. These peptides have since been
found with a high degree of conservation in other crustacean species using both biochemical and
mass spectrometric techniques [54, 57, 59, 102, 195-197]. A recent genomics study used the
translated open reading frames of P. clarkii transcripts as queries in searching the available EST
database of H. americanus. This led to the identification of five ESTs encoding H. americanus
orcokinin orthologs. Further analysis led to the discovery of two distinct prepro-hormones, that,
when processed, would generate 13 orcokinin or orcokinin related peptides, several of which
were verified in vivo from MS analysis of tissues [198]. Many isoforms of orcokinins have been
discovered or predicted in crustacean species.
55
It was not until recently that orcokinins were found in insects. The first insect orcokininlike peptide was found in brain extract from the cockroach Blattella germanica in 2004.
However, it did not show activity in a gut tissue assay. Further genomic analysis by the same
authors identified putative orcokinin-like peptides in several other invertebrates, including the
mosquito Anopheles gambiae and the nematode Caenorhabditis elegans. Several other insect
species in addition to A. gambiae were investigated but not found to have orcokinin-related
sequences [199]. This finding provided the first evidence that the orcokinin peptide family is
present across several phylla, although it does not appear to be universally present in arthropods.
Immunocytochemical studies have shown that the orcokinin family is present extensively
throughout the nervous system, neuroendocrine structures, and fibers throughout the gut [54,
200, 201]. Orcokinins have also been detected in the hemolymph at an approximate
concentration of 10-11 M using an ELISA assay [193]. This finding suggests that orcokinins may
be released into hemolymph and thus function as hormones in addition to their roles as locally
acting neurotransmitters.
Unlike most invertebrate NPs, the orcokinin peptides are neither N-terminally nor Cterminally blocked, with a few exceptions (C-terminal amidation) observed in crustaceans. This
is atypical but not unique to the orcokinin NP family [202]. Bungart et al. studied the bioactivity
of orcokinin analogues in the hindgut assay, and demonstrated that changes at the C-terminus
have a lower impact than changes at the N-terminus in terms of activity [203].The uniqueness of
a free N-terminus and the results of these analogue assays indicate that this portion of the
orcokinin sequence is vital to its biological activity.
Despite the extensive biochemical characterization of the orcokinin family, relatively
little is known of their physiological roles. As mentioned previously, the bioactivity of the
56
orcokinins was first demonstrated in the hindgut of the crayfish [192, 200]. In addition, synthetic
orcokinin has been applied to the STNS of Homarus americanus, with an alteration in the pyloric
rhythm observed [54]. An additional putative role for orcokinin peptides is in circadian rhythms.
In the cockroach Leucophaea maderae, the accessory medulla (AMe)—a small neuropil in the
optic lobe believed to contain the master circadian clock—has orcokinin-like immunoreactivity
[204]. Phase shifts in circadian locomotor activity were observed in the cockroach after injection
of Asn(13)-orcokinin near the AMe at different points in the animal’s circadian cycle [205].
Functional roles for orcokinins include modulation of the central pattern generators of the
crustacean STNS and regulation of the circadian clock in the cockroach.
2.6.5. Crustacean Cardioactive Peptide (CCAP)
Crustacean cardioactive peptide (CCAP) was initially purified from the pericardial organs
of the shore crab Carcinus maenas [206]. This peptide has 9 residues and an amidated Cterminus. It also has a disulfide bridge (PFcNAFTGc-NH2, with c indicating a Cys residue that
participates in a disulfide bond). These structural characteristics, in particular the disulfide bond,
are rarely found in crustacean NPs. After its original identification in C. maenas, CCAP was
identified in several other crustaceans [59, 101] with a conserved sequence. CCAP
immunoreactivity is present throughout the nervous system in crustaceans, but is found at a
higher concentration in the pericardial organ [207-209].
2.6.5.1. Functions of CCAP in the Crustacean
CCAP modulates the heartbeat in a number of crustacean species [152, 206, 210, 211],
likely by acting on the cardiac ganglion (CG). The CG is a central pattern generator (CPG)
located within the single chamber heart. The CG is a well described CPG, containing five motor
neurons, and four pacemaker interneurons. These motor neurons are electrically coupled, which
57
leads to synchronous firing to produce heart contraction [212, 213]. Investigation of exogenous
CCAP on the intact working heart, on the semi-intact heart, and on the isolated CG shows that
CCAP released from the POs as a neurohormone regulates the crab heart by acting on the heart
muscle itself at lower hemolymph CCAP concentrations and on the neurons of the CG, most
likely the premotor interneurons, at higher concentrations. Depending on the concentration (and
thus the cell type affected), CCAP modulates contraction amplitude and frequency, as well as
burst amplitude, duration, frequency, and number of spikes per burst [211].
Several other non-cardiac roles for CCAP have been discovered. In the horseshoe crab,
Limulus polyphemus, CCAP was found to act on the midgut [208]. It also has activity in the eye,
decreasing the electroretinogram amplitude, and thus the light sensitivity, in the isolated retina of
the crayfish Orconectes limosus [214]. Several electrophysiological studies have also showed the
effects of CCAP on the crab STNS [215, 216]. Work by Phlippen and colleagues used an
immunoassay to determine the CCAP content of hemolymph in O. limosus and C. maenas
undergoing ecdysis. CCAP levels were greatly elevated during the active phases of ecdysis—
delineated as the stages in which the shell is actually shed, relying upon much movement,
including rhythmic muscle movements—as opposed to the passive phases, which are
characterized by increased water intake to swell the body to enable shell removal. They thus
postulate that CCAP regulates several physiological changes observed in crustaceans around the
time of molt, including heart rate and STNS activity changes, in addition to behavioral and
muscle activity changes necessary for molting [217]. Although the primary described role of
CCAP is on the heart itself, other functions have been observed. It is possible that CCAP
coordinates these functions to occur at the same time as alterations in the heart rate as part of
important processes in the animals’ life cycle, such as molting.
58
2.6.5.2. CCAP in Other Invertebrates
In several insect species, CCAP or peptides with very similar sequences have been
isolated and characterized [218, 219]. Many of these CCAP peptides have been discovered from
genetic information. In Manduca sexta, the CCAP gene was isolated and determined to encode
125 amino acid residues in a prepropeptide that contains a single copy of CCAP. This CCAP
gene is very similar to the CCAP gene found in Drosophila melanogaster [220]. As observed in
crustaceans, CCAP causes contraction of the insect heart when applied in vitro [221-223]. Direct
activity is also observed on a variety of muscles, and it follows the pattern of other
neurotransmitters/neuromodulators. One such example is its activity on the leg muscle of M.
sexta; CCAP excites rhythmic contractions [221]. It has also been observed to stimulate hindgut
contraction in this species [219, 224]. Furthermore, CCAP can act upon the smooth muscle of the
locust oviduct. A dose-dependent increase in contractions was observed, and oviducts of locusts
at earlier developmental stages were found to be more sensitive to this effect. Spontaneous and
neurally-evoked contractions were both effected [225, 226]. In addition, CCAP-like
immunoreactivity has been detected in other smooth muscle neurons, including those in the
abdominal ganglion that project to the spermatheca of adult female locusts. The study that
identified this immunoreactivity also assessed the effect of CCAP on contractions of the
spermatheca, and CCAP increased the basal tonus and frequency of spontaneous contractions, as
well as the amplitude of neurally evoked contractions [227]. These data suggest that CCAP is
likely to play an important role in reproduction via its activity on smooth muscles in reproductive
organs. An additional role for CCAP has been identified in the cockroach, Periplaneta
americana, for which the activity of digestive enzymes following food ingestion was increased
by CCAP. The authors of that article postulate a system in which CCAP is released by gut
59
endocrine cells in response to food intake, and CCAP alters the efficacy of digestive enzymes
[228]. This direct modulation of enzymatic activity is an uncommon mode of action for NPs.
A putative CCAP GPCR—CG6111—has been identified in Drosophila melanogaster.
This GPCR has sequence homology to the arginine vasopressin (AVP) receptor in vertebrates
[229]. Although it is homologous to the AVP receptor, CG6111 is activated by adipokinetic
hormone (AKH) and CCAP but not AVP. One CCAP GPCR was cloned from the mosquito
Anopheles gambiae, and this receptor was found to be related to receptors for AKH and
corazonin [230]. CCAP exists in insects as well as crustaceans, and a great deal of similarity in
sequence, localization, function, and receptor type is observed between the two groups.
2.6.6. Corazonin
2.6.6.1. Insect Corazonins
Corazonin was first identified from the nervous system and hemolymph of insects. A
number of different isoforms of corazonin exist with slight sequence variations. Many of the
isoforms appear to have species-specific activities. [Arg7]-Corazonin (pETFQYSRGWTNa) was
first isolated from the corpora cardiaca (CC) of the cockroach Periplaneta americana, in which it
is a potent cardiac stimulator [231]. The [Arg7] isoform has also been found in numerous other
insect species. Another isoform—[His7]-corazonin (pETFQYSHGWNa)—was later isolated
from the CC of the locust Schistocerca americana. This [His7]-corazonin also activated the
Periplaneta heart muscle [232]. Significnatly, neither isoform altered the heart beat in the locust.
The precursor of a third corazonin—[Thr4, His7]-corazonin (pQTFTYSHGWTNa)—was
recently cloned from the honey bee Apis mellifera, and the final active form was verified by
MS/MS [233]. Additional application of MS techniques to corazonin discovery in insects has
yielded three additional isoforms—[Gln10]-corazonin (pQTFQYSRGWQNa), [Tyr3, Gln7,
60
Gln10]-corazonin (pQTYQYSQGWQNa), and [His4, Gln7]-corazonin (pQTFHYSQGWTNa)—
from members of Mantophasmatodea and Hymenoptera [234]. [Arg7]-Corazonin was originally
described from P. americana but is found in many other insect species. In contrast, the other
isoforms are restricted to only a few insect groups or even single species. It is therefore
postulated that corazonin modifications appeared late in the evolution of insects [234]. Several
corazonin isoforms exist in insects, with some being more widespread than others.
Like the presence of [Arg7]-corazinin, the presence of corazonin in the neurosecretory
neurons of the CNS of insects seems to be highly conserved [235-240]. Other
neurosecretory/neurohemal organs in P. americana contain corazonin, including the head
neurohemal system in the retrocerebral complex, and in neurosecretory cells in the brain [34].
These neurosecretory cells project across the glandular part of the CC to reach their release sites.
Corazonin has also been identified in the hemolymph of P. americana by immunological
methods, which suggests it has a hormonal role [238]. Hormonal function is also supported by
the long half-life of [His 7]-corazonin in hemolymph—determined to be 30 min by HPLC and
MS analysis. This contrasts with the short half-lives of other neuropeptides, which usually
degrade in a few minutes [241]. CCAP localization is highly conserved among insect species,
and, along with its presence in the hemolymph and relatively long half-life, this is indicative of a
hormonal role.
2.6.6.2. Crustacean Corazonin
[Arg7]-Corazonin is the only corazonin isoform that has been identified in crustaceans. It
was first found in the pericardial organ (PO) of the crab Cancer borealis using MALDI-TOF,
and its sequence was confirmed by PSD fragmentation and by comparison with a synthesized
corazonin standard [55]. It was later found in several other crustacean species with a conserved
61
primary sequence [57, 102]. In addition to the PO, it is also present in the CNS and STNS. Its
release can be stimulated from the STG both by electrical stimulation of projection nerves and
depolarization by K+ [172]. The isoform of corazonin found ubiquitously in insect species is also
the isoform found in crustaceans. It is widely distributed throughout the nervous systems of
crabs.
2.6.6.3. Functions of Corazonins
Corazonin has a variety of functions, most of which have been studied in detail in insects.
It can potently activate the isolated hyperneural muscle and the antennal heart of P. americana,
but is not active on the oviduct or proctodeum [240]. These effects of corazonin on visceral
muscles appear to occur only in Periplaneta species, as muscles in other cockroach species and
the heart of Schistocecra americana (as mentioned above) showed little or no sensitivity to
corazonin. In addition to its myotropic effects in certain insect species, [His7]-corazonin was
found to induce the development of a black pattern in locusts in response to crowding [242]. A
similar phenomenon was also observed in the grasshopper Oedipoda miniata, where injection of
[His7]-corazonin caused darkening of the integument in a dose-dependent manner [243].
Corazonin’s effect on integument color has also been observed in the crayfish Procambarus
clarkii. An interesting additional function for corazonin is revealed by experiments that
demonstrated behavioral changes induced by corazonin in Locusta migratoria, Schistocerca
gregaria, and Bombyx mori. In particular, it causes these insects to swarm and interact, as
opposed to being solitary. The same researchers also found that corazonin changes thorax
morphology in these species [244-248]. A final postulated function for corazonin is in the
process of ecdysis in the hawk moth. Researchers have demonstrated that corazonin stimulate
secdysis-related hormone release from the Inka cells in Manduca sexta [249]. Corazonin has an
62
important role in regulation of muscle excitability, particularly in the heart of Periplaneta
cockroaches. It also alters the color of several insects and one crustacean species, although it is
not classified with the canonical pigment-altering hormones. It seems to affect behavior and
morphology in some insects, and promote ecdysis in one identified species. Many of corazonin’s
functions are restricted to certain species, and a general role for corazonin in insects has yet to be
determined. [250]. The function of corazonin in crustaceans remains largely a mystery.
2.6.6.4. The Corazonin Receptor
Very little is known about the mechanism by which corazonin acts on target cells to
produce the results described above. A corazonin-sensitive GPCR has been identified from the
genome of Drosophila. It has a high degree of homology to the AKH receptor [229, 251]. A
corazonin-sensitive GPCR has similarly been identified from A. gambiae. The Anopheles
gambiae corazonin receptor is also related to its AKH receptor and its CCAP receptor [230].
Although very little is known about the molecular targets of corazonins, putative receptors have
been identified from Drosophila and A. gambiae.
2.6.7. SIFamides
The identification of the first SIFamide peptide was achieved in 1996 by extraction from
350,000 grey flesh flies (Neobellieria bullata) followed by extensive HPLC purification and a
combination of ESI-QTOF MS and Edman degradation for amino acid analysis [252]. The Cterminal SIFamide sequence is the identifying characteristic for NPs of this family. Currently, six
different isoforms are known from both insects and crustaceans, with high sequence conservation
among these species.
Like many NPs, the first function discovered for SIFamide was myoactivity. It was
observed that SIFamide potently activated the L. migratoria oviduct, but it was found to have no
63
effect on the hindgut [252, 253]. Eventually, targeted cell ablation and RNAi revealed that
SIFamide modulates sexual behavior in the fruit fly in a dramatic manner, with flies lacking
SIFamide being highly promiscuous [254]. However, very few other studies have been
performed to determine the functions of SIFamides in insects. Verleyen et al. recently reviewed
several aspects of this peptide family, including progress in identification, localization, and
assignment of physiological functions, as well as reviewing the discovery of the SIFamide
GPCRs [255].
Originally, crustacean SIFamides were grouped with FMRFamide-related peptides. AntiFMRFamide antibody was observed to react with Penaeus monodon eyestalk extract, prompting
further searches for FaRPs in this organ. The eyestalk extract was then analyzed by MALDITOF and Edman degradation to reveal the presence of a SIFamide with primary sequence of
GYRKPPFNGSIFamide along with six FaRPs [99]. Later, another SIFamide was found in the
lobster Homarus americanus with a sequence of VYRKPPFNGSIFamide [256]. This compound
differs only on the first position of the N terminus, and is sometimes called [Val1]-SIFamide
compared to [Gly1]-SIFamide. These two SIFamide sequences have since been observed in
several other crustacean species, although [Val1]-SIFamide has only been discovered in
Homarus spp. and C. maenas [102, 175]. An additional putative peptide with the sequence
RKPPFNGSIFamide has been observed in MS analysis of neural tissue extract of several
crustacean species [57, 59, 102]; however, it is not clear if this fragment exists in vivo or is
present due to degradation. Its activity has also not been tested. Mining of the Daphnia pulex
genome has yielded a putative SIFamide with sequence TRKLPFNGSIFamide. A peak with
mass matching the theoretical mass for this compound was identified by MS but MS/MS
sequence verification could not be conducted [86]. The nomenclature of this peptide is a bit
64
tricky, as it is shortened by one AA at the N-terminal end and has two substitutions—a Thr for a
Tyr at the N-terminus and a Leu for the Pro at its fourth residue. MALDI imaging and
immunohistochemistry studies have shown that SIFamide is widely distributed in the crustacean
nervous system, including both the CNS and STNS [28, 256].
Christie et al. reported that [Gly1]-SIFamide is present in the midgut epithelial endocrine
cells in several Cancer species,and is released upon exposure to high-potassium saline [257],
which points at a possible hormonal role for SIFamide. Application of synthetic [Val1]SIFamide to the stomatogastric ganglion (STG) increases the amplitude and duration of bursts in
the pyloric dilator (PD) neurons, causing an activation of the characteristic pyloric pattern [256].
Both [Val1]- and [Gly1]-SIFamides possess an internal dibasic site, [Arg3-Lys4], which could
possibly undergo cleavage during prohormone processing to lead to the isoform
PPFNGSIFamide. However, this octapeptide has not been detected via MS analysis of any of the
investigated species, and did not produce significant activity changes when applied to the
isolated STNS of H. americanus [175].
2.6.8. RYamides
RYamides were first found in the pericardial organ (PO) and PO releaseate (after high-K+
depolarization) from Cancer borealis [55]. Five different isoforms were sequenced in the
original study, all possessing RYamide on the C terminus with higher variability at the N
terminus. Most of these isoforms along with additional RYamides were later found in other
crustacean species, such as Cancer productus [101] and Carcinus maenas [102]. Relatively little
is known about the RYamides. Their localization has been mapped by MSI to the anterior bar
region of the PO in C. borealis, a pattern not common to other NPs investigated in the study,
including A-ASTs and RFamides [13]. In addition to the PO, some RYamide isoforms have also
65
been detected with lower abundance in the brain in several crab species [59]; however none of
these peptides have been seen in the lobster H. americanus [57]. When applied to the isolated
crab STG, RYamides did not produce overt physiological effects [55]. Therefore, the study of
this peptide in other tissues will be required to understand the physiological functions of this
peptide.
2.6.9. The CHH/MIH/GIH family
The crustacean hyperglycemic hormone (CHH) superfamily is one of the largest groups
of neuropeptides identified in crustaceans. Members of this family have been identified in more
than thirty crustacean species (Table 2). Typically, these peptides are produced and released
from the X organ- sinus gland (XO-SG) complex in the eyestalk, and are known to regulate
numerous essential biological processes, including carbohydrate metabolism, reproduction, and
molting. CHH-family peptides are also among the largest known crustacean neuropeptides, with
mature peptides from this family having a molecular mass of 8–9 kDa. Another conserved
feature is the presence of six cysteine residues at similar positions in their sequences, with the
potential to form three disulfide bonds. The CHH family is divided into type I and type II
peptides, based on their primary structures. The best studied peptides in this family are CHHs,
which belong to the type I subfamily. Homology above 60% is observed in the primary
sequences of type-I CHHs between different species. The type II subfamily contains NPs with
different sequence characteristics from the type I peptides. These type II peptides have a variety
of different functions, among them regulation of molting (molt-inhibiting hormone, MIH),
reproduction (gonad-inhibiting hormone, GIH, also known as vitellogenesis-inhibiting hormone,
VIH), and the varied functions of the mandibular organ (mandibular organ-inhibiting hormone,
MOIH). Biochemical and genetic techniques have identified several precursor sequences for the
66
different CHH subtypes. These preprohormones of CHH-family peptides also contain CHH
precursor-related peptides (CPRPs), which are located between the signal peptide and the actual
hormone. The CPRPs have not yet been studied intensively. The CHH/MIH/GIH family is one of
the most extensively studied neuropeptide families in crustaceans. Several review papers have
been published discussing their structures, localizations and physiological functions [258-264].
Here the authors will update some more recent findings.
2.6.9.1. CHH in the Crustacean
The CHH/MIH/GIH family is large and growing, having gained several new members in
the last few years, due mostly to the expansion of genetic information available. These peptides
are the largest known crustacean peptide hormones with molecular weights (MWs) between
8000 to 9000 Da, compared to 500-2000 Da for other neuropeptides. This larger size is a main
reason that conventional characterization of CHH peptides relies on Edman degradation and/or
cDNA cloning as opposed to MS-based techniques. Smaller crustacean NPs have MWs in the
region for which mass spectrometers have had excellent performance over the last several
decades, whereas only recently have high-resolution MS instruments been developed with the
capability to adequately characterize compounds with MWs in the mass range of the CHHfamily peptides. Use of these high-resolution mass spectrometers in conjunction with a suite of
tools typically used to study proteins has recently been reported by Ma et al. (2009). These
researchers developed a MS technique combing both top-down (intact molecule fragmentation
MS/MS) and bottom-up (enzymatic digestion of the peptide prior to MS analyses of each
resulting fragment via MS/MS) strategies to characterize a novel CHH isoform from the crab C.
borealis. No previous work had identified a CHH sequence solely with MS-based methods [56].
CHH-type I peptides have been more extensively studied since their first isolation and
67
identification from the XO-SG complex in 1979 [265]. This isoform was later found in other
tissues by antibody-based techniques. For example, Keller et al. employed RIA to detect CHH at
low concentrations in the pericardial organs of C. maenas [266]. Other immunochemical
localization studies have identified CHH immunoreactivity in the second roots of the thoracic
ganglia of H. americanus [267], the retina of P. clarkii [268], and the gut and abdominal
peripheral neurons of C. maenas [269, 270]. The CHH family is one of the best-studied NP
families in crustaceans, although MS-based study of it has been limited until recently. Its
localization throughout several organs of the crustacean nervous system has been described using
immunochemical techniques.
2.6.9.2. CHH Function in Crustaceans
CHH is an important hormone that possesses multiple functions in crustaceans
throughout all the life stages of the animal. As its name implies, CHH type I peptides cause
hyperglycemia and hyperlipidemia in the hemolymph as part of the crustacean’s regulation of
energy metabolism and storage [260]. Through binding with hemocyanin, CHH travels from
neuroendocrine organs to the target tissues, such as midgut and musculature, through the
hemolymph. It is thought that hemocyanin protects it from degradation by enzymes in the
hemolymph, much as albumin does for peptide hormones in mammals. At the target tissues,
CHH causes the release of free glucose from glycogen stores in a manner reminiscent of
glucagon. CHH can also induce breakdown of lipid stores in the midgut, thus increasing
hemolymph fatty acid levels [271, 272]. In addition to the hyperglycemic role of CHH, it is
involved in other processes, such as osmoregulation [273], control of ecdysis [269, 274],
inhibition of ovarian protein synthesis [275, 276], and inhibition of androgenic gland function
[277]. It appears that the role of type I peptides is primarily to act on distant target tissue,
68
including endocrine glands, with a variety of end functions. Some of these functions overlap with
those of the type II (GIH, MOIH, MIH) subfamily, such as action on ecdysis and reproductive
organs.
The adaptive character of CHHs has also been extensively studied. The level of
hemolymph CHH increases in response to changes in environmental conditions such as oxygen,
temperature, salinity, parasitism, heavy metals and exposure to lipopolysaccharide (causing
immune system challenge) [278-283]. It should be noted that CHHs from the pericardial organ
have different primary structures and possibly different functions compared to CHHs released
from the SG. This is illustrated in research conducted by Chung and Zmora [278]. These authors
looked into the expression and release of SG-CHH and PO-CHH in C. sapidus in response to
stress induced by hypoxia, emersion and temperature change, concurrent with monitoring
hemolymph glucose and lactate levels. Hemolymph glucose and lactate levels increased
dramatically under all of these stresses. Glucose levels also significantly increased when SGCHH was injected into crabs, but PO-CHH did not have the same effect. In addition, hypoxia
caused significant changes in the hemolymph concentrations of SG-CHH but not PO-CHH
[278]. This suggests that SG-CHH carries out the more traditionally understood role for CHH,
that of energy regulation, particularly in response to environmental stress. PO-CHH does not
appear to be involved in these processes, and thus has a currently unknown function. Other
functional studies of CHH in the crustacean are similar to this one in that they rely upon
immunochemical methods for detection. They typically implicate CHH type I peptides in the role
for which they were named, energy metabolism, in response to a variety of factors.
69
2.6.9.3. MIH in the Crustacean
MIH is a member of the CHH/MIH/GIH type II subfamily, and it too has been found in
many decapod species. In a recent review, MIH peptides were discussed in detail [262]. At the
time of that review, 26 MIH or MIH-like sequences were known, coming from varied decapod
species. The receptor for MIH has been identified, although it is not fully characterized. This
receptoris localized primarily in the Y-organ. From in vitro studies, it has been determined that
MIH delays molting by suppressing ecdysteroid synthesis by the Y-organs. A number of other
features of MIH peptides are also discussed in detail in the review [262]. In summary, MIH
peptides are type II peptides of the CHH family that are widely distributed among decapods.
They inhibit molting, as their name implies, by acting directly on the Y-organ’s synthesis of
ecdysteroids through a receptor about which little is known.
2.6.9.4. GIH in the Crustacean
Compared with CHH and MIH, only a small number of gonad-inhibiting hormones
(GIHs) have been characterized to date. The first peptide with in vivo GIH activity was isolated
from the American lobster Homarus americanus [284]. The mRNA encoding GIH-like peptide
has also been found in the Norway lobster Nephrops norvegicus [285] and the shrimp Penaeus
monodon [286]. Like other CHH family peptides, they are present in the SG-XO complex. The
GIH transcript has also been identified from other tissues, such as the brain and thoracic and
abdominal nerve cords in the shrimp [286]. As its name may imply, GIH is a vital peptide
hormone in crustacean reproduction. Its inhibition of gonad maturation is achieved by
modulating the synthesis of vitellogenin (Vg), which is a precursor for yolk proteins [287]. This
is illustrated by a study in which knockdown of Pem-GIH induced by double-stranded RNA
increased the transcript level of Vg. Therefore, GIH inhibits Vg at the gene expression level
70
[286]. Although GIH is not as well studied as CHH type I peptides and MIH, its function is
understood to be inhibition of gonad maturation and formation of yolk proteins.
2.6.9.5. MOIH in the Crusteacean
Another member of the type II CHH subfamily is mandibular organ-inhibiting hormone
(MOIH). Mandibular organs (MOs) have a homologous role to the corpora allata in insects. This
organ is essential in reproduction and development. The corpora allata is regulated by
allatostatins in insects, but these peptides share no sequence similarity to MOIH. Discovery and
functional studies of MOIH have been carried out in several crustacean species. In the crab
Cancer pagurus, two MOIH peptides were identified, and in an ex vivo assay of isolated MOs,
both inhibited synthesis of the terpenoid methyl farnesoate (MF) [288]. A study in the crab
Libinia emarginata identified a peptide with a CHH-like sequence. Investigation of its function
showed that this peptide inhibits MF synthesisand has hyperglycemic activity, thus giving this
peptide a putative assignment as a CHH-family type II peptide similar to MOIH [289]. The
inhibition of MF synthesis by MOIH has also been investigated in vivo, although the results do
not conclusively verify this function for MOIH. The effects of injecting eyestalk-ablated male
Cancer pagurus with synthetic MOIH-1 or MOIH-2 were compared with injection of sinus gland
extracts. SG extracts reduced hemolymph levels of MF by 60-80%; however, injection of
MOIH-1 or -2 did not dramatically affect MF levels. Treating the sinus gland extracts with
protease abolished their activity, which indicates that an additional peptide or protein in the
extract—other than MOIH—may be responsible for the in vivo activity of the sinus gland extract
[290]. It is also possible that both peptides must be present for activity. Although ex vivo studies
suggest that MOIH inhibits MF synthesis by the MO, in vivo studies provide a more complex
71
picture of MF synthesis regulation. Thus, regulation of the mandibular organ may require
coordination of multiple hormones produced by the SG.
2.6.10. Pigment-Dispersing Hormone (PDH)
Pigment-dispersing hormones (PDH) were discovered along with pigment concentrating
hormones (RPCH) in early neuroendocrine studies of the crustacean eyestalk, and these two
families are sometimes grouped into a single superfamily [4]. Although the peptides have similar
canonical functions—acting on the distribution of pigment granules in specialized colorcontaining cells, they share little sequence similarity and act on different subtypes of cells. For
this reason, these two families will be treated separately in this review.
Initially called distal retinal pigment hormone (DRPH), PDH was discovered after
investigations into the molecular entities that caused migration of pigments in the retinae of
crustaceans, as reviewed by a number of authors [4, 291-294]. The first PDH family
neuropeptide was sequenced in 1976 as NSGMINSILGIPRVMTEA-amide from the shrimp
Pandalus borealis [295]. A different PDH isoform was later found in the crabs Uca pugilator
[296] and Cancer magister [297], with the sequence NSELINSILGPRVMTEA-amide. These
two types of PDH, designated α and β respectively, were later found in numerous species, with
additional isoforms containing only a few amino acid substitutions also identified (reviewed in
[298] and [299]). Several postulated PDH sequences, including both α and β types,determined
from genomic analysis exist [294], but they are not listed here. As indicated, many more
isoforms of β-PDH than α-PDH have been identified, and β-PDH has been discovered in more
species. Some species also contain multiple isoforms of β-PDH. It was once postulated that only
prawns of the Pandalus genus expressed α-PDH, but it has also been described in
Macrobrachium rosenbergii from cDNA clones [300]. Although there are two different types
72
and many isoforms of each type, the overall sequence similarity is high and all members are
octadecapeptides. Thus the PDH family is considered a highly conserved family among
crustaceans. Differences between the α and β types lie mostly in positions three and 4, which
contain large, charged residues at neutral pH (E, L) in β-PDH but small, neutral residues (S, G)
in α-PDH. The full significance of these differences is not known. When a species has multiple
isoforms of one of the PDH types, they typically vary in only a few amino acids, and these
changes typically do not drastically alter the overall properties of the molecule. The presence of
multiple isoforms in a single species may indicate intricate regulation of physiological processes,
or simply redundancy.
2.6.10.1. Distribution of PDH in Crustacean Tissues
PDH localization has recently been described in detail in a review [294]. It is found
mostly in the cerebral ganglion (contains eyestalks and brain) and ventral ganglia of crustaceans.
The eyestalks are considered to be the lateral part of the protocerebrum, although they may be
separated from the rest of the brain by long fiber tracts. These tissues demonstrate a great deal of
PDH immunoreactivity in several species, with PDH-reactive cell bodies in the more medial
parts whose fibers terminate in the sinus gland—located more laterally, and in the medial
protocerebrum and other more medial ganglia. In the medial protocerebrum, immunoreactivity is
observed in cell bodies in an area of neuropil with an identified role in photoreception. in the
medial protocerebrum, Most of the immunoreactivity for PDH in the brain is in neuropil with
cell bodies originating in the eyestalk, but there are a few paired cell bodies that contain PDH
that send projections to the circumesophageal ganglion (COG). Differential distribution of
isoforms of β-PDH has also been observed, with the general finding that Cancer productus βPDH II is the only isoform found in the SG, and thus it is likely a neurohormone while isoform I
73
is more likely to be a local neurotransmitter [294]. Overall, the distribution of PDH isoforms in
the cerebral ganglion (including eyestalk) suggests both local activity, with projections typically
originating from cells in the eyestalk, and a neurohormonal role, as many fibers terminate in the
secretory structure SG.
Also described in the review is localization of PDH in other structures. Outside of the
cerebral ganglion, PDH-immunoreactive fibers are found in the ventral nerve cord, COG, and
stomatogastric ganglion (STG). They are typically found in neuropil and as fiber terminals,
although some species have somata in the COGs that stain for PDH [294]. This localization
pattern is more suggestive of a neurotransmitter role for PDH, although its presence in structures
far from the eyestalk may indicate that it is mediating circadian effects on ganglia not directly
involved in adaptation to light. Overall, the distribution of PDH in crustacean ganglia suggests
that it is not simply involved in adaptation of the retinal pigments to light changes. Its presence
in a neurosecretory organ and projections and cell bodies that are far from the eyestalk point to
additional roles, possibly in using light signals as a trigger for other physiological changes.
2.6.10.2. Function of PDH in Crustaceans
The primary function of PDH is translocation of pigment granules in chromatophore cells
by moving them toward the distal parts of these stellate cells. This has been reviewed by many
authors [4, 294]. Certainly, the effect of PDH on isolated chromatophores is well established:
application of it or crude extracts containing it cause pigment cells to express color, as the
pigment granules are dispersed centripetally [4]. This color change is of importance for day/night
adaptation and camouflage. One genus in which circadian color change has been well-studied is
the fiddler crab Uca, which undergoes a darkening of pigment during the day and a lightening
during the night, with the darker pigment in the daytime acting as protection from the light of the
74
sun. This pattern of pigment migration based on time occurs even in the absence of light, and
two other crab species have been shown to have a similarly persistent time-related color change
[294]. Migration of pigments in retinal pigment cells is also a main function of PDH, as it allows
adaptation of the eye to low or high ambient light conditions. It also affects the electrical activity
of photoreceptor cells, mediating changes in sensitivity that are important for vision at different
times of the day [4, 299]. PDH has been shown to cause pigment dispersion in chromatophores
and retinal pigment cells. In general, it helps the crustacean adapt to high ambient light
conditions by this mechanism.
The possibility of PDH as a circadian hormone in crustaceans has been studied in detail
due to a number of factors. First, the persistence of circadian color change in the absence of an
external stimuluspoints to an internal clock, and the ability of PDH to cause color change
suggests it may be part of that system [4]. Secondly, the related pigment dispersing factors
(PDFs) in insects have well-established roles in circadian rhythms, mostly as output messengers
that relay time-related signals from the central nervous system’s clock circuits to target sites [4,
294]. Few studies have directly shown that the concentration of PDH varies in a circadian
manner [301]. However, one recent study found increases in the levels of PDH immunoreactivity
in the brain photoreceptor neuropils (BPNs) of Procambarus clarkii and Cherax destructor that
could be correlated with the animals’ distinct circadian patterns of locomotor activity [302]. In
general, they saw increased PDH immunoreactivity during and immediately preceding periods of
increased locomotor activity, although the two species differ in their exact patterns of circadian
activity. Significantly, PDH is expressed at the beginning of the dark photoperiod, in
contradiction to its role in pigment dispersion in chromatophores. Pigment concentration, not
dispersal, should occur during the dark period. This also correlates with a period of increased
75
activity in many crustaceans, including both crayfish species in this study. However, P. clarkii
also has a period of increased activity near the end of the dark photoperiod, and in this species,
PDH immunoreactivity was also increased during this time. For C. destructor, which does not
exhibit activity around the time of the dark-light transition, PDH immunoreactivity also did not
increase at this time [302]. These findings, in particular the increase in PDH at the beginning of
the dark period, suggest a circadian role for PDH in addition to its chromatophore dispersal role.
Unchanged levels of PDH at the end of the dark period in C. destructor are surprising and
provide further evidence for this. This study suggests that PDH is mediating time-related signals
about the transition from one light phase to another that may signal for increased locomotion.
Recently, Solís-Chagoyán et al. (2012) demonstrated that daily injections of P. clarkii β-PDH
were sufficient to alter the period and phase of a pattern of electrical activity in the retina that
shows a circadian pattern of that species. It also altered spontaneous activity in an area of the
brain that is suggested to form part of the circadian system due to connectivity (the cerebroid
ganglion). Thus, the authors postulated that PDH acts upstream of the retina, on the cerebroid
ganglion, to alter its responsiveness in a circadian manner [303]. This effect, in addition to its
downstream effects on the pigment and photoreceptor cells, is likely a hormonal component of
the circadian rhythm. A great deal of evidence exists to suggest that PDH is involved in the
internal clock of crustaceans. It has been demonstrated to act as an effector of the circadian
clock, as it appears to act on chromatophores and neurons directly, similar to the role of PDFs in
insects.
2.6.10.3. Molecular Action of PDH and its Receptor
The PDH receptor has been identified as a G-protein coupled receptor in Daphnia pulex
via analysis of its homology to C. elegans and D. melanogaster pigment-dispersing factor
76
receptor genes [299]. Although the sequences of PDH and PDF receptor genes are known in
several invertebrate species, little is known about the intracellular signaling components of the
PDF/PDH response. Pharmacological knockout experiments have made some progress in
identifying parts of the signal cascade. As reviewed by Nery and Astrucci in 2002, signaling
through Gs to adenyl cyclase produces an increase in cAMP, which causes activation of cAMPdependent protein kinase (PKA). The authors also point to phosphorylation of an unknown
protein with molecular weight around 58kDa as a possible downstream component of the
signaling pathway [304]. Few studies have addressed the mechanisms by which PDH causes
pigment dispersion in crustaceans, although Gs, adenyl cyclase, and cAMP have been implicated.
Some work has recently been done in Drosophila to dissect the steps of the signaling
pathway through genetic techniques. The PDF receptor in the fly is a Class II GPCR similar to
calcitonin-CGRP receptors. It causes increases in cAMP, likely through Gs. It was also found to
increase intracellular Ca2+ as a second messenger [305]. The cAMP increase has later been
determined to be mediated by adenylate cyclase AC3 in vivo in a subset of Drosophila
pacemaker cells in which the PDF receptor is active. Other GPCRs in the same cells do not
signal through AC3, and it is postulated that a physical association between the PDF receptor and
AC3 is responsible for this specificity of second messenger activity. The A-kinase anchoring
protein nervy is a component of cellular scaffolding that may be at least partially responsible for
this [306]. This has interesting implications in how molecules that can be activated by multiple
upstream signaling proteins in theory are specific to only a few signaling pathways in vivo.
Although it has been studied in greater detail, it is still unclear how PDF causes pigment
dispersion in insect chromatophores. Several of the same intracellular messengers have been
identified as in the crustacean studies.
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2.6.11. Red Pigment Concentrating Hormone (RPCH)/Adipokinetic Hormone (AKH)
Red pigment concentrating hormone (RPCH) was first isolated from the shrimp Pandalus
borealis in 1967 [307]. The full sequence was then determined in 1972 to be
pQLNFSPGWamide [308]. Similar hormones were found in insects, with roles in energy
mobilization, in the adipokinetic hormone (AKH) family. These peptides typically have a
sequence 8-10 amino acids long with several conserved features: pGlu at the N-terminus,
amidation at the C-terminus, Phe or Tyr at position 4, Trp at position 8, Gly at position 9 (if
present), and no net charge at physiological pH [309]. Several reviews have discussed this family
in insects in great detail [202, 291, 292, 309, 310 , 311-313]. In insects, a large amount of variety
is found in the sequences of peptides in the AKH family, but in decapod crustaceans, only one
sequence has been identified—pQLNFSPGWamide—although it has been seen in at least 10
species [309]. A variant form, pQVNFSTSWamide, has been predicted from the ESTs of the
crustaceans Daphnia magna [84] and Daphnia carinata [314], and the genome of Daphnia pulex
[85]. The low sequence variation of this peptide across species and absence of multiple isoforms
is uncommon for a neuropeptide in Crustacea, especially as the insect species display a large and
varied set of AKH peptides.
2.6.11.1 RPCH Distribution in Crustacean Tissues
Red pigment concentrating hormone was first isolated from eyestalks, and its existence in
this tissue has been extensively documented. Only a few of the many studies that have
demonstrated RPCH in the eyestalk are listed here: [307, 311, 315-317]. Localization in the
eyestalk is consistent with the canonical function of RPCH in adaptation of skin coloration to
different environments. Early studies found RPCH in the ventral nerve cord (VNC) and
substances released from the VNC after electrical stimulation [318]. RPCH has also been
78
identified in releasate from the stomatogastric ganglion upon stimulation of several nerves that
have inputs to this ganglion [172], and by immunochemistry in the STG and PO in addition to
the eyestalk sinus gland (SG) [319]. The existence of RPCH outside of the eyestalk and its
release upon nerve stimulation are indicators that it may be released upon multiple stimuli in
addition to alterations in light or color that are perceived in the eyestalk. It may thus represent a
more global signal in the animal, altering multiple pathways in response to light and/or color in
the environment. Although NPs released by the SG can enter the general circulation of the
crustacean, release of RPCH by the PO, VNC, and directly at the STG will have a more direct
and concentrated effect. This may also suggest a role RPCH in crustaceans as whole-body
modulators of energy mobilization, similar to the identified roles of the related adipokinetic
hormones in insects.
2.6.11.2. Function of RPCH in Crustaceans
The function for RPCH determined in early studies of the crustacean was alteration of the
localization of pigment within chromatophore cells, as reviewed by Lars Josefsson, a researcher
largely responsible for the identification and initial sequencing of RPCH from prawns, in 1983
[291]. The author’s interest in RPCH was stimulated by its ability to change the distribution of
pigment in the cells of the integument, which permits some species to change color. This article
illustrates different stages of pigment distribution in a red pigment-containing cell of the
hypodermis (erythropore). In the absence of RPCH, pigment is distributed widely throughout the
star-shaped cell. The effect of RPCH is to move pigment particles within the erythropore toward
the center of the cell, which results in a loss of overall color in the animal. This allows animals to
adapt their coloration to the conditions to which they are exposed. Josefsson also indicates that
crustaceans without eyestalks have dispersed pigment in their erythropores, and injection into a
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de-stalked shrimp became a useful bioassay for the presence of RPCH in the injected substance,
as its injection into these animals led to loss of color (blanching). Additional studies reviewed in
the article demonstrated that RPCH causes concentration of pigment in all types ofthe
hypodermal chromatophore cells found in several other crustacean species—erythrophores (red),
leucophores (white), xanthophores (yellow), and melanophores (brown/black)—although not all
types in all species were responsive [291]. This article provides a comprehensive summary of the
role of RPCH in altering the chromatophore cells in the hypodermis of crustaceans. The
canonical function of RPCH described in this article is to concentrate pigment molecules at the
centers of chromatophores, with effects seen in several crustacean species and many (but not all)
types of chromatophores—concentrating not just red pigments, but white, yellow, and
brown/black pigments as well. It is also clear from this article that the majority of RPCH that is
responsible for these color changes comes from the eyestalk of the animal. Additional reviews
have summarized the role of RPCH in alteration of pigment distribution in chromatophores [5,
292, 293, 320].
As color change has long been associated with ambient light conditions [321], it is
possible that RPCH constitutes a molecular signal of light intensity, which may indicate a role in
altering other processes that are different during day and night and throughout seasons. Its
pigment concentrating effects have been observed in the distal retinal pigment cells of the
crayfish Procambarus clarkii [322, 323]. RPCH not only changes the distribution of pigments in
the retinal cells of P. clarkii, but it also increases the electroretinogram (ERG—a measure of the
sensitivity of the retinal cells to light) in vivo in both light- and dark- adapted animals [323] and
in isolated retinas (Orconectes limosus) [214]. This direct effect on the retinal cells’
responsiveness to light, independent of the location of the pigment, suggests that RPCH has an
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effect on the activity of the photoreceptors themselves. The level of RPCH in the eyestalk of P.
clarkii is also seasonally variable [324]. Thus, circadian and seasonal modulation of visual
perception in crustaceans is another function of RPCH.
Although the primary function of the related adipokinetic hormones in insects is energy
regulation, and some studies have found that crustacean RPCHs can induce such changes when
introduced to insects [88], RPCH has not been observed to have this role in crustacean studies. A
compound with sequence identical to the Pandalus borealis RPCH was identified in the isopod
Porcellio scaber, and found to induce hyperglycemia without affecting physical activity level
when applied topically to these insects [325]. A potential role in energy mobilization is
suggested by the activity of RPCH in the stomatogastric nervous system [172, 326-333], which is
primarily responsible for movements of the crustacean stomach. In this system, a number of
different neuromodulators including RPCH can induce an inward current in the lateral pyloric
(LP) neuron with an amplitude that depends on the cell’s resting potential, resulting in increased
excitability of this neuron and as a result, the rest of the STG circuit [331]. Each of these NPs
leads to a different STG output and affects a different subset of cells; for RPCH the cells altered
are the LP and anterior burst (AB) neurons [332]. Changes in this circuit are not only in
activation of certain patterns, but also in modulation of these patterns. Movement of the stomach
leads to digestion, which can increase hemolymph glucose levels. The ability of RPCH to cause
this in a system that is also regulated by a large number of other neuromodulators suggests that it
is part of a mechanism that integrates information from various modalities to produce different
outputs. However, inducing hyperglycemia by increasing digestion is a vastly different
mechanism than the energy mobilization from stores induced by AKH family hormones in
insects. The link between RPCH and stored energy mobilization in crustaceans has not yet been
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established; however, RPCH is one member of a set of neuromodulators that together provide
information to the STG to elicit stomach contraction, which does have the overall effect of
increasing available energy.
RPCH has also been shown to increase activity in several other crustacean ganglia, and at
the neuromuscular junction. The crayfish swimmerets are controlled by a set of neurons in the
abdominal ganglia that form pattern-generating circuits. The patterns of output changedin
response to RPCH, slowing the swimmeret movement pattern by increasing the length of motor
neuron bursts and the period between them [334]. RPCH increased the amplitude of muscle
contraction in a simple preparation, likely by increasing the release of the primary
neurotransmitter by the motorneuron [335]. It also has been shown to activate and modulate the
cardiac sac rhythm and cardiac ganglion (CG) in several crustacean species, leading in general to
patterns that would increase heart rate and contraction strength [152, 328, 329]. It can also
activate different rhythms depending on the location within a network where it is applied, can
cause different networks to synchronize, and make two networks join together, as reviewed in
2001 [336]. In these studies, RPCH has been shown to have diverse modulatory actions. The
amplitude of firing is increased, and the timing of bursts is altered differentially depending on the
system. They are increased in the CG and neuromuscular junction, but decreased in the crayfish
swimmeret system. Overall, it appears that RPCH increases the tendency of crustacean neurons
in these systems to fire, which is consistent with observations in the STNS.
2.6.11.3. Molecular Action of RPCH and its Receptor
Like most NPs, the receptor for RPCH is expected to be a G-protein coupled receptor
(GPCR), although it has not yet been identified in a published work. A putative Daphnia pulex
RPCH receptor is present in GenBank [337], with accession no. EU503126, submitted in 2008
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by Hamers et al. [338]. This receptor was identified by homology to insect AKH receptors, many
of which have been published by the same research group [230, 251, 339-341]. Further research
and sequencing of additional crustacean genomes may lead to a more confident identification.
The mechanism by which binding to this receptor produces pigment aggregation in
chromatophores has been studied in detail, but is still not completely understood. A number of
works were recently compiled into a model by Milograna et al. that explains the process of how
RPCH binding to its receptor leads to translocation of pigment granules along the cytoskeleton
[342]. Following binding to its receptor, the typical heterotrimeric G-protein signal transduction
pathway is activated. This somehow leads to activation of ryanodine receptors on the
intracellular smooth endoplasmic reticulum, which causes release of Ca2+ through channels in an
unknown mechanism. This increased intracellular Ca2+ leads to closure of the K+ channels that
normally leak K+ when the cell is at rest, which is then balanced by the Na+/K+ ATP-ase. The
mechanism by which the increased intracellular Ca2+ leads to closure of these channels is also
unknown, although the authors postulate it could be due to the change in Ca2+ concentration
itself, a secondary action of the G-protein subunits activated by the RPCH receptor, or
phosphorylation by protein kinase C/Ca2+-calmodulin. Closing this channel causes the cell to
depolarize greatly, which leads to opening of several types of Ca2+ channels (voltage-gated and
others) on the cell surface. This causes extracellular Ca2+ to enter the cell, and once it reaches a
threshold concentration, it activates Rho-protein kinase directly and/or through production of
cGMP. This protein kinase phosphorylates a specific myosin II molecular motor, which is
attached to a pigment granule. The myosin then moves the pigment granule toward the center of
the cell [342]. This mechanism may also be responsible for the other functions of RPCH in nonchromatophore cells. The presence or absence of RPCH, leading to efflux of intracellular
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calcium, could lead to circadian or seasonal alterations in other roles beyond color change. This
mechanism would also lead to increased excitability of neurons with RPCH receptors by
depolarizing their resting potential; this is consistent with observed evidence that RPCH
increases activity in the crustacean in the retina, in several pattern-generating circuits, and at the
neuromuscular junction.
The mechanisms by which the various insect AKH family hormones exert their metabolic
effects have also been studied in detail and reviewed [343]. As mentioned previously, the AHK
receptor is a GPCR. It also causes release of Ca2+ from intracellular stores and influx of
extracellular stores, although the mechanism is different and better understood. A variety of
signaling pathways are employed in insect cells to produce mobilization of a several different
stored energy compounds, actuated largely by increases in intracellular Ca2+ and activation of
phosphorylases that break down the molecules used for energy storage. These signaling
pathways include adenyl cyclase activation of phosphorylase b kinase, phospholipase C (PLC)induced release of Ca2+ from the endoplasmic reticulum via inositol triphosphase, and PLC
activation of protein kinase C by diacyl glycerol leading to phosphorylase activation [343].
Although the intricate mechanisms have been simplified here, the overall effect of AKH binding
to its GPCR in insect cells is to metabolize stored energy molecules, with cAMP, PLC, and Ca 2+
as mediators. Such a large variety of intracellular signaling pathways for AKH family peptides in
insects permits a wide variety of specific signals to be transmitted, as AKH is known to initiate
metabolism of three different types of stored energy molecules. Most of the pathways discovered
in insects are upstream of the known pathways for RPCH signaling in crustaceans, and it is
possible that further study into the “missing links” in the RPCH signaling chain may identify the
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same messengers that are employed by AKH, in particular those that lead to release of Ca 2+from
intracellular stores.
2.6.12. Pryokinins
Peptides of the pyrokinin family were first discovered in insects, where their regulation of
reproduction is well-characterized in several species. For information about these peptides in
insects, recent review articles are suggested [344, 345]. For several aspects of pyrokinin
peptides, information only exists in the context of insect systems; they will not be discussed here
(pyrokinin receptor, reproductive function, molecular action, interaction with other factors).
Insect pyrokinins are defined by having a common C-terminal motif of FXPRLamide,
with X as T, S, G, or V [344]. In crustaceans, the common FXPRLamide motif is also observed,
although additional amino acids can occur in the X position. The N-terminus typically adds an
additional 2 to 5 AAs. For Penaeus vannamei, the first crustacean in which a pyrokinin was
identified, S and N occupy this position in the two identified pyrokinins [346]. Homarus
americanus has S in the X position in its one identified pyrokinin [57]. In Litopenaeus vannamei,
L and N are observed in addition to S [347]. The amino acids A and S occupy this position in the
pyrokinins of Carcinus maenas [102] and Cancer borealis [59]. Finally, in Callinectes sapidus,
the observed pyrokinin has an S in this position [348]. In the single crustacean with a sequenced
genome, Daphnia pulex, pyrokinins were not identified, likely due to the short sequence tag that
could be used for interrogation of genetic sequences [85-87]. In Litopenaeus vannamei, a
pyrokinin could also not be identified from the known genomic information that was available
for that species [347]. It is possible that transcriptomics strategies are not successful in finding
sequences with homology tags of this size. Sequences of pyrokinins are characterized by the
FXPRLamide motif in insects and crustaceans, although instead of having T, S, G, or V
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occupying the X position, N, S, A, and L are observed in crustaceans. It is clear that flexibility of
the sequence at this position is a common theme among invertebrates, and although the exact
AAs observed in crustaceans can differ from those in insects (with the exception of S), many
have similar properties on the side chains. G and A are typically classified together as non-polar,
small AAs. V and L are considered to be medium-sized, hydrophobic AAs. N and T, however,
do not have many similar characteristics. The ability to have different pyrokinins with similar but
distinct receptor properties due to slight alterations in sequence is an advantage possible in both
insect and crustacean pyrokinins.
The localization of pyrokinins in invertebrates suggests multiple modes of action, both
directly on identified neural circuits and hormonally through circulating fluid. In insects,
pyrokinins are found in the brain-subesophageal ganglion complex, corpora cardiaca, ventral
nerve cord (VNC), and abdominal and thoracic ganglia [345]. In crustaceans, they have been
identified in cell bodies in the commisural ganglia (CoGs), and neuropil of the STG and CoGs of
C. borealis [59, 65]. In this species and C. sapidus, it was found in the pericardial organs (POs)
as well [59, 65, 348]. Pyrokinin-family peptides were found in the brain and VNC of H.
americanus [57] and L. vannamei [347], and the brain of C. maenas [102]. It was also isolated
from an extract of the entire central nervous system of P vannamei [346]. This wide pattern of
distribution does not suggest a single role for pyrokinin peptides. The presence within the STNS
circuits suggests a role in stomach contraction. PO localization suggests a hormonal role.
Presence in the central nervous system indicates a more complex function. No single role is
suggested by the patterns of localization of pyrokinin peptides in crustaceans, and thus local
neurotransmitter and hormonal roles are both likely.
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The primary roles of pyrokinin peptides in insects are regulation of reproduction and gut
motility, although additional diverse functions have recently been suggested, including
regulation of diapause and coloration [344]. Far less is known about pyrokinin function in
crustaceans. Only a few studies have investigated its action in the well-characterized circuits of
the crustacean STNS, using Cancer borealis. It was determined to activate the gastric mill
rhythm, acting on all neurons involved in this rhythm, with all 4 pyrokinin peptides having
essentially the same effect [65]. It was later shown that output of the gastric mill rhythm
following pyrokinin application was nearly identical to that produced by activation of a single
identified STG neuron, MCN1. This is achieved in an interesting manner, by changing the
relationships of neurons within the circuit to one another. The same neurons are employed by the
MCN1-stimulated gastric mill rhythm as by the pryokinin-stimulated gastric mill rhythm, but the
way in which their firing affects each other is altered [349]. The significance of this finding is as
yet unknown in the overall physiology of the crab; however it has importance in understanding
neuronal circuit organization and modification by neuropeptides. Pyrokinin clearly has an
important role in the crustacean STNS, eliciting a vital rhythm for digestion. Another study
found that it was decreased in the brain after feeding in C. borealis via MS-based stable isotopelabeling quantitative methods [350]. This is indicative of a release of stored pyrokinin,
degradation of extracellular pyrokinin present in the unfed state, reduced synthesis of pyrokinin,
or a combination of these factors. It is possible that neurons from the CoG, where cell bodies are
positive for pyrokinin, are releasing that substance into the STG or other areas to stimulate the
gastric rhythm, instead of into the brain. Other roles are also likely, based on localization of the
peptide and functions of pyrokinins in insects.
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2.6.13. Proctolin
2.6.13.1. Proctolin Discovery and Sequence Identification in Arthropods
In 1967, a substance, first termed “gut-factor,” was isolated from the gut of the American
cockroach Periplaneta americana [351]. This substance was found to stimulate visceral muscle
contractions, and could not be identified as any of the known insect neurotransmitters at the time.
Proctolin, as the compound was later known, then was isolated and sequenced (RYLPT) in 1975,
followed by its synthesis and description of its function on the cockroach hindgut [352] in an
assay that later became a standard preparation for assessing putative NP activity, the hindgut
assay [353]. Since its discovery and identification, proctolin has been extensively studied in
insects, focusing on its distribution among different species, structure-function relationships, and
role in feeding behavior (more recent reviews: [108, 344, 354, 355]). In insects, it acts as a cotransmitter to modulate the postsynaptic response to another NT. This action occurs at the
neuromuscular junction, synapses in the central nervous system, and distant receptors in an
endocrine manner (reviewed in [354, 356]).
After its discovery in P. americana, proctolin was subsequently applied to several
different preparations of lobster ganglia and motor neuron synapses, and changes in activity were
observed [357-359]. Endogenous proctolin in the crustacean was later demonstrated by a study in
the crayfish Procambarus clarkii by a number of methods, including that a component of a
muscle extract was chromatographically indistinguishable from [3H]-proctolin [360]. Schwartz
and co-workers later verified its presence in an extract from Homarus americanus by fast-atom
bombardment MS with a mass measurement error of 2.8 ppm [361]. The authentic sequence of
proctolin as RYLPT from crustacean tissue was first obtained by Stangier and co-workers in
1986 using Edman degradation [362]. Subsequent studies have demonstrated that this sequence
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is identical in many insect and crustacean species [363]. A comprehensive review of proctolin’s
identification, distribution, function, and related substances in crustaceans does not exist; and a
comprehensive review is beyond the scope of this article. The following section will focus on
identification and distribution of proctolin in crustacean species. Its function on neural circuits
and muscles is complex, and will not be covered in detail. Other aspects of its biological
functions will be described, along with some information on its molecular action.
2.6.13.2. Identification of Proctolin in Crustacean Species
Proctolin’s sequence has been positively identified as RYLPT in several crustacean
species, using Edman degradation, tandem MS, and/or transcriptomics. Following Edman
sequencing of authentic proctolin as RYLPT in Carcinus maenas [362], a number of studies
confidently verified the existence of proctolin with an identical sequence in other crustacean
species. Proctolin’s presence was confirmed by mass spectrometry in Cancer borealis first by Li
et al. in 2003 [55], with further identification in this species in a later work [59]. Other studies
went on to identify the peptide RYLPT in Homarus americanus [197], Cancer productus [101],
Carcinus maenas [102], and Callinectes sapidus [364] by MS/MS. Tandem MS has been a
powerful tool for identification of proctolin across many crustacean species. One study identified
both proctolin and [Met-4]-proctolin via MS/MS and genome mining in Daphnia pulex.The
MS/MS spectrum for RYLMT was of lower quality, and it has not been seen in insects [86]. The
lower quality MS/MS spectrum may indicate a lower abundance of this alternative form. A
variety of other genomics studies have met with variable success in identifying proctolin in
crustaceans. Searching of D. pulex ESTs in 2009 was unable to identify the sequence of
proctolin, but this is likely due to the poor performance of the software for identifying small
peptides from genomic information as well as the limited amount of genomic information
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available at the time, as the authors observed proctolin-like immunoreactivity [87]. Another
study also failed to find RYPLT in EST databases for a number of crustacean species in 2008
[84]. Later studies of D. pulex after the genome became available in 2010 (at wfleabase.org),
identified the sequences RYLPT and SSWGVDARYLPT, along with the proctolin precursorrelated peptide SDPLSPIGPPRGEDP [85]. EST mining was sufficient to identify proctolin in
Litopenaeus vannamei, although the same publication failed to find it via MS [347]. The variable
success of genome and EST mining for proctolin demonstrates the importance of a
comprehensive database and difficulty when using tools designed to identify large sequences for
finding NP sequences, which are typically much shorter. In fact, identification of the proctolin
genes was greatly aided by the discovery of the preproproctolin gene in Drosophila
melanogaster [365], which provided a larger sequence for searching in some of the studies noted
here. Proctolin is found in many crustacean species with the same sequence, RYLPT, and one
variant has been observed and verified by both genomics and MS/MS. This high degree of
sequence conservation across Crustacea, and indeed Arthropoda, is unusual for a NP and may
indicate that this NP is highly important and may exist in more species than previously
postulated. The identification of a variant by genome mining and its verification by MS/MS in
the one decapod crustacean with a sequenced genome hints, however, that previous studies
identifying proctolin as RYLPT may be incomplete. Mining the genome permitted identification
of the variant without relying on MS/MS, which has a bias for identification of the highest
abundance compounds unless targeted methods are used. Further genomic analysis may provide
sequences of putative variant forms, which could be verified by targeted tandem MS.
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2.6.13.3. Distribution of Protcolin
In crustaceans, proctolin is widely distributed across several ganglia. Early studies in H.
americanus identified proctolin-like immunoreactivity in nearly every portion of the nervous
system [361]. The distribution of proctolin in the nervous systems of several decapod crustacean
species has been described in detail. Although there is some inter-species variation in proctolin
distribution [209, 366-369], the overall patterns are similar. In general, studies have found the
most intense protctolin immunoreactivity in neurosecretory cells, mainly in synaptic varicosities.
The neurosecretory organs PO and SG have high proctolin concentrations, primarily in axonal
areas [209, 319, 361, 362, 366, 368-372]. The proctolin-containing cell bodies that project to the
POs are found in the esophageal ganglia (OG) and brain, and other proctolin-positive cell bodies
in the OG project to the commissural ganglia (CoG) and stomatogastric ganglia (STG) [209,
366-368, 371, 373-377]. Protcolin neurons are present in the thoracic and abdominal ganglia,
from where they project to other ganglia and/or have a neurosecretory role in function of the
hindgut or postural stances [360, 368, 371, 378]. Several motorneurons with proctolin content
have been identified, and its role in these neurons appears to be similar to that observed at the
insect motor synapse—as a cotransmitter that modulates the response to other transmitters [368,
371, 373]. The STG contains proctolin primarily in neuropil [369, 379]. Finally, proctolin is
found in somatosensory/mechanosensory cells of the thoracic and abdominal ganglia [368, 371,
373]. The localization of proctolin within the crustacean nervous system can be generalized as
follows: nearly every ganglion contains proctolin, and it is present mainly in cell bodies and
axons. Some ganglia contain proctolin primarily in cell bodies (esophageal ganglia,
thoracic/abdominal ganglia), and some have it mostly in axonal projections from other areas
(pericardial organ, sinus gland, stomatogastric ganglion), but few contain both (brain,
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commissural ganglia). Axonal projections appear to take the form of either synaptic varicosities
(“beads-on-a-string”) or discrete synaptic bouton-like structures. This pattern of localization
suggests that proctolin has two main functions: neurohormonal via release into the hemolymph
to affect distant targets, and neurotransmission via direct projection between ganglia. Most
ganglia either send or receive proctolin signals, not both; thus each ganglion may be a site for
integration of multiple peptide signals. Although it is nearly ubiquitous in the crustacean nervous
system, the varied distribution of proctolin across different ganglia is indicative of its roles as
neurohormone and neurotransmitter.
2.6.13.4. Proctolin Function on Neural Circuits
By far, the most research into proctolin’s function has gone into analysis of its effect on
central pattern generators (CPGs), neural circuits that fire in rhythmic patterns, typically with an
output on muscle movement. Main CPGs in crustaceans include the stomatogastric nervous
system, responsible for gut and stomach movements; the cardiac ganglion, responsible for heart
movements; some motor neuron circuits involved in walking and posture; and neurons in control
of gill ventilation. In general, proctolin has the effect of increasing the activity of a neural circuit
[380]. This increased activity could occur at any number of levels in the circuit, includingsensory
input neurons that initiate a rhythm or monitor its efficacy, interneurons that integrate signals
from multiple other neurons, motor neurons that signal the output to muscles, and muscles
themselves. Proctolin may act at multiple levels, and may affect different levels in different
circuits. For a more detailed description of its action in several circuits and at the motor neuron
synapse, a review article is suggested [380].
The sensitivity of some somatosensory neurons has been shown to be increased by
proctolin, specifically in control of ventilation and walking legs. This was first demonstrated by
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Pasztor and Bush in 1987 [381]. These same authors later found that proctolin was released after
stimulation of stretch-sensitive neurons in the oval organ, which is important for regulation of
gill ventilation [382]. Further experiments demonstrated that proctolin’s augmentation of the
sensitivity of somatosensory neurons was opposite to the effect of the classical neurotransmitters
serotonin and octopamine [383]. Thus, the authors postulated that local release of proctolin by
the oval organ increases the sensitivity of that organ’s mechanosensory inputs, which in turn
causes more proctolin release. This positive feedback loop can also be attenuated by
monoamines, and the balance between proctolin and classical neurotransmitters permits
regulation of the ventilation rate. A similar function was observed in the stretch receptors of the
walking legs in crayfish: sensitization of the mechanosensitive neurons [384]. In contrast,
proctolin was found to have no effect on the anterior gastric receptor (AGR), a mechanosensory
neuron that receives inputs from stomach muscles, although the AGR was sensitive to a
FMRFamide-like peptide. It should be noted that the AGR is located just outside of the STG
[385]. Thus proctolin modulates neural circuit activity in some cases by increasing the sensitivity
ofsomatosensory neurons.
In central pattern generating circuits (CPGs), proctolin has the ability to initiate certain
patterns and/or modulate them. It has been studied in the context of nearly all identified
crustacean CPGs in several species, starting soon after its discovery. In the cardiac ganglion
(CG), proctolin has been shown to initiate the cardiac sac rhythm and increase the frequency and
duration of spike bursts, as well as the spike frequency and number in each burst [152, 328]. It
also acts on nearly all cell types involved in the pyloric rhythm (one output component of the
crustacean STNS), and is able to induce this rhythm and modulate several features of the pattern
[332]. This is part of how proctolin and other NPs can produce similar currents in a specific cell
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of the STNS, via activation of a number of different cells in the network, but lead to different
overall circuit outputs [331]. Modulation of the pyloric rhythmis postulated to form the basis for
changes in the gastric rhythm (the other output of the STNS) elicited by proctolin, as these two
rhythms are distinct yet connected, and proctolin appears to have little effect on most of the cells
that form this rhythm [386]. Proctolin has also been studied in the context of crustacean
swimmeret [387-390] and leg [391] movements. It was found to typically cause excitation of
swimmeret movements, acting alone as a neurotransmitter in the pattern-generating neurons of
this system, although this effect occurs only in a subset of the motor neurons [387]. In leg
movements, it had a different effect depending on the neurotransmitters with which it was
applied [391]. When applied with dopamine or concurrent with activation of descending positive
inputs, it led to a leg movement associated with a courtship display. Octopamine was able to
prevent proctolin from inducing this behavior, unless dopamine was not also added [391]. In
crustaceans, proctolin primarily increases the activity of cells within defined pattern-generating
circuits. This is particularly strong in the cardiac ganglion, pyloric rhythm, and leg/swimmeret
neurons. It many of these circuits, application of proctolin is sufficient for initiation of the
circuit’s characteristic firing pattern, while in others, co-transmitters are necessary. Proctolin’s
action in these circuits demonstrates how signals from diverse peptides can lead to similar but
distinct outputs by acting on a subset of cells in a neural network or working differently
depending on the other transmitters present. Its general tendency to lead to activation of firing in
neurons of these circuits is consistent with the overall identification of proctolin as an activating
peptide.
Proctolin can also act directly on muscles to increase the output of circuits in crustaceans
and insects (mentioned previously). Not surprisingly, its effect on the muscles of the hindgut has
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been investigated in a number of crustacean studies, a few of which will be discussed here.
Proctolin causes different types of contractions in muscles of the hindgut: in the long muscles
that run parallel to the gut itself (longitudinal muscles), it increases the frequency and strength of
contractions that occur spontaneously, and in the ring-shaped muscle fibers that surround the gut
(circular muscles), it causes a slow, long-lasting contraction [392]. Similar studies on lobster
arterial muscles demonstrated that proctolin induces contractions of these ring-shaped muscles,
which leads to decreased arterial diameter and increased resistance to blood flow [393]. Some
studies have found that proctolin’s ability to increase muscle function is due in part to increased
neurotransmitter release in the presence of proctolin at the neuromuscular junction [335, 394],
while others suggest that proctolin changes properties of the post-synaptic cell, permitting it to
fire more strongly and with less stimulation [395]. Whether the activation is pre- or postsynaptic, proctolin acts directly on muscle, likely at the neuromuscular junction, to induce
contractions and increase their strength. By increasing activity at all levels of a circuit—input,
interneuron, and output—proctolin has an overall excitatory effect in the crustacean.
2.6.13.5. Molecular Action of Proctolin
A GPCR in Daphnia pulex has been proposed as the receptor for proctolin by homology
to insect proctolin receptors [2]. Much more is known about the effects of proctolin on targets
further downstream in the signaling pathway. Several studies have indicated that calcium
channels are integral to proctolin signaling [394, 396, 397]. Secondary messengers postulated to
be responsible for this action on Ca2+ channels include cyclic AMP or GMP [395, 398, 399],
protein kinase C (PKC) [399], protein kinase A (PKA) [395], and depolarization by K+ channels
[395]. It is possible that different phosphonucleotides and different protein kinases act in
different crustacean species. It has also been postulated, however, similar to the action of several
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other NPs, that this GPCR leads to Ca2+ release from endoplasmic reticulum, and this, with or
without additional secondary messengers, leads to changes in the electrical activity of the
postsynaptic cell that predispose it to greater intensity and/or frequency of firing, and/or change
the phosphorylation state of muscle proteins, leading directly to their activation [395, 399, 400].
This wide variety of possible sites where proctolin may influence firing and/or contraction
permits it to have a range of functions, from slight alterations in circuit dynamics to dramatic
changes in the animal.
2.7. Conclusions and Future Perspectives
Invertebrate model animals have made significant contributions to the study of
neuropeptides, both in methodology and in understanding how NPs regulate important processes.
Mass spectrometry-based techniques are optimal for studying NPs, due to their high specificity
when compared with other techniques. MS-based tools can generate peptide sequence
information without tany a priori knowledge from a variety of neural tissue samples, ranging
from intact tissues to microdialysate. Mass spectrometry imaging (MSI) is an emerging
technique to map the distribution of NPs in a given tissue with greater specificity than antibodybased imaging techniques and the ability to characterize the distribution of thousands of analytes
in a single experiment. Direct tissue imaging allows rapid identification of NPs from a tissue of
interest, avoiding sample preparation steps that might lead to alterations in the chemical identity
of NPs. Liquid samples can be subjected to a number of analytical separation techniques prior to
MS analysis for improved confidence of identification. Quantitative methods are also rapidly
expanding for MS-based platforms. Assays to determine the function of NPs can gain from the
advances in MS techniques described above. One great advance is in the application of
microdialysis sampling on alert, behaving animals for NP collection and correlation with
96
activity. MSI is also able to improve function determination by localizing NPs to neurons or
subsections of ganglia with known physiological roles. In the age of readily available genome
sequencing, additional tools to predict and identify NPs are available, and the sequencing of the
first crustacean genome, from Daphnia pulex, has greatly increased these capabilities. Many NP
families are known, and many of these have been characterized both chemically and
functionally.
Although recent advances in MS and genomic technology have been highly useful to the
field of invertebrate (and vertebrate) neuropeptide discovery and functional identification, further
technological advances are under development and the full suite of crustacean NPs has not been
described fully. Advances in MS technology will greatly improve the capabilities of this
technique to provide tissue localization, identity, and quantity information. In the area of MSI,
methods for tissue coating with matrix are being improved to create smaller, more uniform
crystals, to decrease analyte diffusion, provide better spatial resolution, increase sensitivity, and
reduce “hot spots” of signal on the tissue. Improvements to laser technology will also improve
the spatial resolution of this technique. For identification, faster scan rates, higher accuracy, and
better resolution will improve the quality of identifications and allow less abundant compounds
to be identified. Similarly, for quantification, increases in MS instrument speed, accuracy, and
resolution along with the development of novel isotopic labeling techniques will enable more
reproducible quantitation of lower concentration analytes. With increased coverage of crustacean
genomes by next-generation sequencing, identification by database search software will likely
improve in the future. Computer software and hardware advances have also increased the ability
to run complex software, including de novo sequencing software, in more reasonable periods of
time. Finally, many crustacean NPs currently have no known function, and a two-stage method
97
for function determination will be of use. First, NPs can be monitored using MS for
comprehensive analysis of all compounds potentially involved in a particular function, especially
with the aid of the minimally invasive sampling technique of microdialysis. This will narrow
down the list of hundreds of NPs to a few “interesting” NPs in an unbiased and systematic
manner. Then, those peptides that appear to be of import for that role can be investigated in
detail, using targeted MS techniques or more traditional techniques, for a more confident
determination of the role of the NPs in that function. All of these advances arepossible due to the
incorporation of MS into the NP discovery and functional identification workflow. Further
improvements to the analysis methods and increased study of NP functions will greatly increase
our understanding of this important class of neurotransmitters, both in crustaceans and in other
organisms, including mammals.
2.8. Respective Contributions Statement
All authors researched, wrote, and edited the paper.
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2.10. Figures
116
Figure 2.1. Neuropeptide synthesis, processing and storage. Neuropeptides are synthesized in
the cell body by the rough endoplasmic reticulum (RER) and packaged into vesicles as
prepropeptides. These are transported down the axon to the terminal, where they are stored until
released. During this transport and storage period, the prepropeptides are subjected to a number
of processing steps, including cleavage and various types of post-translational modification. In
crustacean NPs, conversion of a C-terminal glycine residue into a C-terminal amidation is a
common modification that is illustrated here. After release from the axon terminal, NPs diffuse
across the synapse to the dendrite, where they come into contact with their receptors. NP
degradation occurs following receptor internalization (not shown), or after NPs diffuse to
degradation enzymes, which are present in the synapse and in the extracellular space.
117
Figure 2.2. Overview of MS-based methods for neuropeptide discovery in invertebrates.
Illustrates extraction of tissue, direct tissue profiling, and mass spectrometry imaging (MSI), and
how these methods fit into a workflow. Tissue from an animal can be extracted and subjected to
cleanup and analytical separations techniques prior to MS and/or MS/MS analysis to determine
NP identity. Direct tissue analysis can quickly give the NP profile of a tissue of interest. MSI can
determine the complex distribution of a NP throughout a tissue of interest. The brain of a
decapod crustacean is used in the figure for illustration.
118
Figure 2.3. Workflow for sample preparation options for analysis of NPs in liquid samples.
Liquid NP samples can be obtained via extraction and protein precipitation of dissected tissue or
hemolymph, or from microdialysis. These samples can then be subjected to any number of
solution-phase sample preparation techniques, including immunoprecipitation, desalting, and
chemical modification. One useful type of chemical modification that can be done at this step is
attachment of isotope-coded tags to peptides from different time points or different conditions
for quantitative MS analysis. These reduced complexity and/or modified samples can then be
subjected to analytical separation techniques, two of which are illustrated here. These samples
can then be subjected to MS and/or MS/MS. As indicated by the arrows, some steps can be
skipped or repeated depending on the sample and desired analysis.
119
Figure 2.4. Physiological recording techniques to determine NP function in the crustacean
stomatogastric nervous system (STNS). One method to determine NP function is to apply NPs to
an isolated STNS preparation, which is kept alive in a dish and subjected to extracellular and/or
intracellular recordings using sharp electrodes. a) Illustrates the isolated STNS and sites where
electrical recordings can be made. b) Changes are observed in the stereotypical firing pattern of
the LP neuron and from the lvn nerve when a NP, CbAST-C2 is bath applied to the preparation.
Adapted from [64] with permission. Copyright 2009, Elsevier.
120
2.11. Table
Table 2.1: Detection of NPs in Hemolymph with Different Sample Preparation Methods
Sample Preparation Method
Peptide
Theoretical In vivo In vitro Hemolymph
Peptide sequence
Classification
(M+H)
MD
MD
extraction
A-type
YAFGLa
569.31
x
x
allatostatin
YSFGLa
585.3
x
x
SPYAFGLa
753.39
x
DPYAFGLa
781.39
x
PDMYAFGLa
912.43
x
pERPYSFGLa
949.49
x
ERPYSGLa
967.5
x
B-type
NWNKFQGSWa
1165.55
x
allatostatin
TSWFKFQGSWa
1182.57
x
NNNWSKFQGSWa
1366.63
x
Allatostatin
DPYAFGLGKRPADL 1519.79
x
combination
DPYAFGLGKRPADL
2128.09
x
YEFGLa
TachykininAPSGFQa
605.3
x
related
GFLGMRa
679.37
x
APSGFLGMRa
934.49
x
x
APSGFLGM(O)Ra
950.49
x
TPSGFLGMRa
964.5
x
Orcokinin
NFDEIDRSGFG
1256.55
x
x
NFDEIDRSGFGF
1403.62
x
NFDEIDRSGFGFV
1502.69
x
NFDEIDRSGFGFN
1517.67
x
NFDEIDRSSFGFV
1532.7
x
NFDEIDRSGFGFH
1540.68
x
RFamide
RSFLRFa
824.49
x
RNFLRFa
851.5
x
GGRNFLRFa
965.54
x
DRNFLRFa
966.53
x
SGRNFLRFa
995.55
x
GPRNFLRFa
1005.57
x
APRNFLRFa
1019.59
x
x
SDRNFLRFa
1053.56
x
TNRNFLRFa
1066.59
x
121
RYamide
Other NP
Cryptocyanin
fragment
b-Actin
fragment
DGGRNFLRFa
GAHKNYLRFa
SMPSLRLRFa
SENRNFLRFa
FVGGSRYa
SGFYANRYa
pEGFYSQRYa
pQLNFSPGWa
(RPCH)
PFCNAFTGCa
(CCAP)
TNFAFSPRLa
(CabPK-I)
I/LNFTHKFa
1080.57
1104.61
1105.63
1181.62
784.41
976.46
1030.51
x
x
930.45
x
956.38
x
1051.57
x
YKIFEPL
909.51
YKIFEPLR
YKIFEPLRE
YKIFEPLRES
KIFEPLREDNL
YKIFEPLRESN
YKIFEPLRESNL
1065.61
1194.65
1281.68
1373.74
1395.73
1508.81
LRVAPEESPVL
1209.68
x
x
905.5
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Total NPs
35
8
11
Total protein fragments
2
0
6
Table 2.1 was created from data compiled from references [67-69]. An x indicates that the
compound was detected with that sample preparation method, a blank cell indicates that it was
not. In vivo MD indicates experiments where a microdialysis probe was implanted into a live
crab and sample was collected from this probe for analysis preceded by sample preparation steps.
In vitro MD experiments entailed immersing a MD probe into a beaker of hemolymph freshly
collected from a crab, kept on ice, and constantly stirred. The liquid that flowed out of this probe
was prepared and analyzed. Hemolymph extraction was conducted by adding the hemolymph
immediately to an acidified methanol solution with or without protease inhibitors added,
122
followed by sample preparation and analysis. Analysis was conducted by capLC-ESI-QTOF,
nanoLC-ESI-QTOF, MALDI-FTICR, and/or MALDI-TOFTOF.
123
Chapter 3: Methods for Function-Driven Discovery and Functional Assessment of
Neuropeptides
Adapted from Schmerberg, C. M. and Li, L. Protein Pept. Lett. 2012, May 23. (epub ahead of
print) and Schmerberg, C. M., Chen, R., and Li, L. Prog. Neurobiol. 2012 [in preparation].
3.1. Abstract
A number of unique challenges are inherent to the study of neuropeptides (NPs), both in
determining their molecular structure and their function. Traditional studies follow a model in
which novel NPs are discovered and identified, then investigated for function. These studies
frequently use biochemical techniques that can be imprecise and cumbersome. Mass
spectrometry (MS)-based tools are becoming important not only in precisely determining the
identity of a NP or quantifying a compound with a known sequence, but also in studies where
identity and putative function can be determined simultaneously. Tools based on MS and tandem
MS (MS/MS) have been developed, both with isotope labeling strategies and label-free methods,
that allow accurate quantitation of NP changes associated with behavior or physiological
manipulation, concurrent with identification of sequence. MS and MS/MS have also been
implemented with sampling methods that incorporate temporal or spatial information while
determining functional role of a NP, such as microdialysis (MD) and imaging mass spectrometry
(IMS). These advances in MS and sampling techniques allow investigation of a particular
biological phenomenon to guide studies aimed to identify and characterize NPs. Permitting
function to drive identification of relevant compounds allows for a broader understanding of the
molecular underpinnings of these events. The NPs thus identified can then be validated with
more conventional techniques, and successive iterations of identification and function
124
determination will provide rich information about these compounds. This function-driven
discovery of NPs using MS-based techniques is an important new approach for their study.
3.2. Introduction
Neuropeptides (NPs) are a class of neural signaling molecules whose study has greatly
expanded in the past several years. The physico-chemical characteristics of NPs relative to
classical neurotransmitters require a new set of analytical tools to be developed for their study,
but these distinct features also provide greater diversity of NP identity and functional specificity.
NPs are short chains of amino acids, typically 4-10 residues in length in invertebrates, or 5-30 in
vertebrates (reviewed in [1]). They are also defined by having the ability to signal between
neurons, or between neurons and somatic targets. The great structural diversity and common
sequence motifs among NPs of the same family allow for each peptide to have distinct and
highly specialized functions as well as functions in common with other family members. This
great structural diversity has led to challenges, however, in the discovery of NPs and elucidation
of their function. NPs are synthesized as prepropeptides in neurons (reviewed in [1]). These long
peptides then undergo a number of processing steps, including cleavage into smaller peptides and
modification of residues, such as pyroglutamation or amidation of the C-terminus. These
processing steps take place as the peptides are packaged into vesicles which are finally sent to
the axon terminal for storage prior to release. After release by the neuron, NPs diffuse to the
receiving cells, either across the synaptic cleft or after transport to a distant site via circulating
fluid. NPs in circulation are commonly protected by binding proteins. The peptides then bind to
their receptors, typically members of the G-protein coupled receptor superfamily, to transduce
the signal to receiving cells.
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3.2.1. Neuropeptide Detection and Sequence Determination by Classical Methods and Mass
Spectrometry
A variety of experimental strategies have been employed to study the identities and
functions of NPs. These encompass both targeted and non-targeted methods, and are illustrated
in Fig. 3.1. Detection of NPs has historically relied heavily upon targeted identification and
quantification, frequently with antibody-based techniques. This is problematic due to the high
degree of cross-reactivity that most NP antibodies have for multiple NPs, particularly those
within the same family. It has recently become clear that most NP families have many members,
and not all of these compounds have the same physiological effects, although antibodies cannot
distinguish between them (reviewed in [1]). Cross-reactivity across NP families is also observed,
especially in the case of NPs with common post-translational modifications, such as C-terminal
amidation. Antibodies raised against the longer mammalian neuropeptides are frequently more
specific owing to greater antigen complexity, although it is often unclear what portion of the
sequence the antibody recognizes. This can lead to confusion when different NP isoforms occur
and may have different function [2]. Determination of the exact identity of the active NP under
this scheme requires purification of a large amount of sample to be subjected to Edman
degradation, and more recently, mass spectrometry (MS) and tandem mass spectrometry
(MS/MS) for sequencing.
With the advent of soft ionization techniques for MS, the study of NPs has become more
feasible and useful (reviewed in [1]). Slight differences in amino acid sequence among NP
isoforms, which can lead to antibody cross-reactivity, create noticeable differences in the massto-charge (m/z) ratio observed by MS. MS/MS analysis provides NP sequence information by
selecting analytes with a certain m/z in the first dimension of MS and fragmenting them into
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smaller parts that are then subjected to MS analysis. Confident identification and sequencing of
NPs directly from small biological samples is thus achieved with MS and MS/MS tools. MSbased analysis has identified thousands of putative NPs from a wide variety of species (reviewed
in [1, 3-7]). However, many studies finding novel NPs by MS can only identify compounds as
putative NPs based on sequence similarity to known NPs and/or the organs from which they are
extracted. These identifications are either targeted to a particular molecular entity—as in
quantification of NPs via antibody or MS-based techniques that require prior knowledge about
the compound—or divorced from functional relevance—as in NP surveys that attempt to
describe the entire NP complement of an organism via MS-based techniques.
3.2.2. Classical Methods of NP Function Determination and New Directions
Determination of NP function with classical targeted methods also typically requires
prior knowledge about its sequence. As mentioned previously, under the classical definition of a
NP, it must have activity on neuronal or target cells. This activity frequently leads to a change in
the electrical potential of a cell or the conductivity of ion channels by which this potential is
established and maintained. Thus the gold standard for NP function determination is targeted
electrophysiological study of isolated organs, neurons, or organ systems upon application of the
NP in question (reviewed in [8]). These living cells are removed from animals and kept alive in a
preparation, while various techniques are used to determine the electrical potential of one or
more cells in the system. The NP is applied to the cells, and changes in the potential are noted.
Studies of this type are able to accurately pinpoint the cellular action of NPs and their function in
small neural circuits, but are difficult to implement—requiring specialized equipment and
expertise—and require a pure sample of known composition. Determination of a NP’s function is
frequently more complex than determining its sequence. These studies are also targeted to
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specific putative NPs and particular functions—functional information is only obtained about the
compounds applied on the electrical circuits investigated. These targeted techniques for
determining NP identity and function provide highly specific detail about compounds for which
a priori knowledge is available. However, by focusing on a single NP or physiological process,
many other important compounds or functions may not be observed. Therefore, there are
advantages to a non-targeted approach. One such alternative strategy for non-targeted MS-based
NP study uses manipulation of processes known to be controlled by NPs concurrent with MS
analysis of putative NPs (examples: [9, 10]), an approach here referred to as function-driven
discovery. In this strategy, all compounds that appear to be altered in the course of the
experimental manipulation can be monitored. Changes in these compounds occurring during the
experiment can later be verified with more established methods for neuropeptide function
validation, such as ex vivo electrophysiology as described above, to confirm its identity as a NP
under the classical definition with greater confidence. An advantage is that the identity of a NP
need not be determined prior to analysis; the use of MS-based tools allows for discovery and
sequence identification of new NPs during the course of the experimental physiological
manipulation, and no important compounds are missed by focusing only on a few target NPs. A
few candidate compounds discovered in this way can then be the subjects of more targeted
investigation with traditional techniques.
Mass spectrometry-based function-driven discovery has expanded in recent years, and it
will be the focus of this chapter. Several studies illustrating the use of many of these tools will be
discussed as examples. Particular attention will be paid to studies of decapod crustacean NPs, as
this is an area in which NP research has made great headway. The crustacean is an excellent
system for NP discovery and functional study, as its nervous system is simple, contains relatively
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large neurons in well-defined circuits, and is rich in NPs (reviewed in [1, 5, 8, 11]). Validation of
the candidate NPs identified in function-driven discovery will also be discussed.
3.3. Function-Driven Discovery of Neuropeptides using Mass Spectrometry
Function-driven discovery of NPs requires the ability to measure and identify putative NP
candidates that are differentially expressed or secreted under experimental manipulation, either
qualitatively or quantitatively. Although MS is a powerful tool for discovery and accurate
identification of NPs in small samples, reliable quantitation using MS can be challenging.
Obtaining samples for MS that are correlated with a particular dynamic behavior is also difficult.
Toward these ends, a variety of innovative strategies for MS-based quantitation and live-animal
sampling have been developed. The MS-based techniques are illustrated in Figure 3.2, and
broadly fall into the category of function-driven non-targeted approaches, meaning that they do
not require a priori information about analytes’ identities. These techniques, and MS used in
conjunction with novel sampling methods, will be discussed in further detail.
3.3.1. Novel Mass Spectrometry Tools for Quantitation in Studies of Neuropeptide
Function
Quantitative analysis by mass spectrometry is complicated by the heterogeneity of
ionization and unpredictable discrimination and suppression effects of complex mixtures.
Several strategies have been developed to address these problems. Internal standards can be
added to samples to account for day-to-day and run-to-run variability. Many of the most reliable
MS-based quantitation methods, including selected reaction monitoring/multiple reaction
monitoring (SRM/MRM), absolute quantitation (AQUA) by addition of isotopically labeled
internal standard, and absolute quantitation calibration curves, fall into the category of targeted
analysis and require prior knowledge of the analyte’s m/z and/or its fragments’ m/z, and these
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methods are time-consuming to develop (reviewed in [7]). Often times, this prior knowledge is
not available in a function-driven discovery study. Of these methods, SRM is more popular for
determining NP concentration change as a means to elucidate function. SRM uses a first
dimension of MS to isolate a single parent ion. It is then fragmented, as with MS/MS to
determine peptide sequence. One daughter ion of this fragmentation is then analyzed by the
second dimension of MS. This permits highly accurate quantitation, but only allows a single
reaction to be monitored. For MRM, multiple parent-to-daughter transitions are monitored and
quantified. The most salient feature of these quantitation modes is that they require prior
knowledge of the analytes’ structure and fragmentation patterns, as well as generation of
calibration curves to determine the relationship between the instrument response and analyte
concentration, and thus can only be used in targeted studies of NP function. For these reasons,
function-driven NP discovery can best be achieved with differential display (DD) and relative
quantitation (RQ), due to the lack of a priori information.
3.3.1.1. Differential Display vs. Relative Quantitation
Differential display (DD) and relative quantitation (RQ) are related and often employ the
same analysis strategies. Relative quantitation describes changes in the quantity of a compound
across different biological samples, often as a fold-change. DD is more often used to describe the
all-or-nothing presence of NPs in some samples but not others. This can be of great importance
in determining function, because NPs present in some conditions but absent in others can be
expected to have an important role in the neurobiology of those conditions. However, since a
fold-change cannot be established, it can be difficult to determine the dose-response effect of
these NPs. DD data is often displayed as pairs of mass spectra, mass spectra with isotopic peak
pairs (described below), Venn diagrams, or tables.
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In comparison, relative quantitation (RQ) describes changes in the quantities of NPs
across biological samples. Quantity changes are usually expressed as a change from baseline, or
fold-change between different states. A variety of MS and MS/MS methods are used in RQ, with
important features being the accuracy and reproducibility of quantitation. RQ data allows for
determination of the dose-response effects of NPs on progressive behavior; for instance,
increases in an anorexigenic NP during feeding may lead to reduced food intake rate, resulting
finally in cessation of eating. This allows a more concrete link to be established between the NP
and its function. RQ data is usually expressed in bar graphs showing fold-change in different
NPs between two states, or line graphs showing fold-change versus time.
3.3.1.2. MS-Based Quantitation Methods for Functional Discovery of NPs
RQ and DD data can be obtained using either MS or MS/MS techniques, and these
methods can further be characterized by whether or not they employ isotopic labeling (label and
label-free). Isotopic labeling is a method to prevent differential MS instrument effects from
occurring in different samples. Different isotopes are incorporated into separate biological
samples, the samples are mixed, and analysis of the mixed sample allows for determination of
NPs present in one condition but not the other (DD) and/or changes in NP concentration (relative
quantitation). Because the analytes from different conditions differ only in isotope composition,
they experience the same instrumental conditions, and the response received for each can be used
as a measure to quantitate them [10].
A number of methods for isotope-based MS quantitation exist. Metabolic labeling of
whole animals is an exciting new field. In a scheme similar to stable isotope labeling in cell
culture (SILAC), animals are raised on an isotope-enriched diet, provided by algae or bacteria
that are grown in media containing a single isotope of a vital element, usually nitrogen-15
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(reviewed in [12]). An additional experimental group is fed the same food source, with the
exception that its food source is raised under normal conditions. Each experimental group can be
subjected to different conditions expected to lead to changes in NPs (or left unperturbed, as
controls), and samples can be collected and combined prior to analysis by MS. To prevent
experimental artifacts due to the metabolic labeling, the groups should be switched. The DD of
NPs across conditions can be determined by looking for the presence or absence of normal and
isotopically-enriched compounds. In addition, relative quantity changes can be determined by
comparing the heights of isotopic peaks on a single MS spectrum or the areas under the curve
(AUCs) for the extracted ion chromatograms (XICs) for the normal and isotopic NPs if a
separation method is coupled to MS, i.e. ultrahigh performance liquid chromatography (UPLC)MS. Although metabolic labeling of whole animals is a powerful tool, it is expensive, limited to
animals that can be raised in the lab, and the global incorporation of isotopic elements into an
animal may lead to different phenotypes. Most notably, high-anxiety mice fed
15
N diets had
greater depression-like behaviors in a recent study [13]. This has profound implications for using
metabolic labeling in studies of NP function.
Other methods of isotopic labeling exist for functional discovery of NPs, including
several where isotopes are incorporated as part of the sample preparation procedure. Several
schemes exist to tag biomolecules with isotopes [14-19]. One scheme that has gained much
success is dimethylation with formaldehyde and deuterium formaldehyde [18, 19]. This reagent
adds two methyl groups to any primary amine in the peptide, such as the N-terminus or lysine
side chain. If deuterium formaldehyde is used, these methyl groups will each contain 2 deuterium
atoms, leading to a total mass difference of 4 Da per primary amine. Deuterium formaldehyde is
relatively inexpensive and the addition of deuteriums to a molecule usually does not greatly alter
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its behavior under analytical separation modes, such as liquid chromatography or capillary
electrophoresis. As with metabolic labeling, samples of different isotopic composition can then
be combined and analyzed concurrently. The ratios of peak heights obtained from a MS
spectrum or ratios of AUCs from the XICs if separation is employed prior to MS can then be
calculated to yield a fold-change in concentration of that analyte. A schematic illustrating this
technique is shown in Fig. 3.2 under the heading “Mass Difference Tags.” This figure illustrates
the pairs or triplets of peaks observed in such a MS spectrum. Most such tools use only two
reagents, but depending on the isotope and label, more reagents may be possible. This technique
has successfully been used to quantitate changes in the NP content of several ganglia of the
decapod crustacean Cancer borealis after feeding [10, 19]. In Fig. 3.3, the mass spectrum
obtained from the extract of brains of fed and unfed crabs is shown [10]. In this work, 12 NPs
had statistically significant (p<0.01) expression level changes after feeding in the brain, and 6
were altered in the pericardial organ (PO). Mass difference tags are a useful tool for global
assessment of relative abundance changes of NPs in small samples.
Label-free methods for MS-based quantitation also exist, including standard addition
(reviewed in [20]). A standard can be added to samples at the same concentration and the
response of analytes relative to that of the ITSD can be determined. This method assumes that
the response of the instrument to the NPs, relative to the response of a standard compound, is
linear in the concentration range over which the analysis is run. With most mass spectrometers
and typical working ranges, this is usually true. This technique is similar to absolute quantitation
by standard addition, but skips the step where a calibration curve for the analyte and internal
standard is created. Since many studies quantify hundreds of NPs at once, and the sequences of
important NPs are not always known prior to analysis, it is impractical to generate calibration
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curves for all analytes prior to analysis and standard addition without calibration can identify
interesting target compounds for further study. For this type of analysis, ITSD (usually a NP
from another species to avoid potential overlap and interference) is added to the sample, the
sample is run on chromatography with MS detection, and software is used to create XICs for
compounds that are abundant and that show NP-typical chromatographic profiles, characterized
by cohesive peak shape and elution around the same time as the NP ITSD. The AUC is
calculated for each and divided by the AUC for the ITSD. The concentration changes of these
compounds can thus be tracked, and those that change in response to the experimental
manipulation can be selected for further study, including determination of the NP sequence. This
technique is illustrated in Fig. 3.2 under the heading “ITSD/AUC,” and is represented by a series
of TICs obtained each of the samples. These TICs would then be extracted and normalized to
allow quantitation of changes in NPs over time. Data obtained via standard addition and MS
analysis must be treated as semi-quantitative and not definitive, but it is useful for the purpose of
functional NP discovery.
3.3.1.3. MS/MS-Based Quantitation Methods for Function-Driven Discovery of NPs
Tandem MS (MS/MS) can also be used for quantitation of NPs as part of a study to
determine their function. MS/MS quantitation techniques have the advantage of providing
sequence information in the same instrumental run as quantitative information, but they lack the
sensitivity of MS-based techniques. As with MS-based techniques, isotopic labels can be
incorporated into samples, or label-free methods can be applied. Tandem mass tags (TMTs) are
reagents used for MS/MS quantitation of compounds, and these include isobaric tagging for
relative and absolute quantitation (iTRAQ), dimethylated leucine-based tags (DiLeu) [19, 21],
and others. A full description of the method by which these tags permit quantitation and
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sequence identification is out of the scope of this dissertation, and a recent review is referred to
for detailed discussion [21]. Briefly, TMTs come in sets of reagents. Each reagent is reacted with
a different sample, and the labeled samples are then mixed. The tag adds the same total mass to
the peptide for each reagent, but upon MS/MS fragmentation, each tag yields a characteristic
low-mass reporter ion due to the incorporation of different numbers of isotopes. The full
complement of peptide fragments are also observed, and thus comparing the peak heights of the
low-mass reporter ions permits relative quantitation and the remaining sequence-specific
fragment peaks allow for sequence identification. A graphical representation of this is shown in
Fig. 3.2 under the heading “Tandem Mass Tags.” This shows the MS/MS spectrum from a TMT
experiment, with fragment ions corresponding to the NP sequence present as well as low mass
reporter ions that correspond to each of four samples. iTRAQ has been used for relative
quantitation of NPs from distinct neurons in the F cluster of the cerebral ganglion of Aplysia
californica [22]. This method is robust and reliable, although, since it relies upon MS/MS; a
larger quantity of peptide must be present compared to MS-only techniques.
Label-free techniques also exist for MS/MS-based RQ of NPs in function-based
discovery studies as well. Spectral counting is a method that determines the relative abundance
of a compound based on the number of MS/MS events for which it is selected. Since ions are
selected for MS/MS fragmentation based on their relative intensity, this method is a reasonable
estimation of relative abundances of corresponding peptides. A number of additional concerns
must be addressed when conducting spectral counting, and these are reviewed in [20]. Data
obtained from spectral counting is often used to generate a profile of how NP concentrations
change relative to each other at each time point, and this is illustrated in Fig. 3.2 under “Spectral
Counting.” Although consecutive runs on a single instrument can be compared for quantitative
135
analysis, especially if the spectral counts can be normalized to a peptide/protein that does not
change across the time points, it is more reliably used to assess changes in the proportions of NPs
that compose each sample. This is shown in the figure by illustrating changes in the relative
amounts of NPs of each of several common crustacean NP families. Spectral counting and TMTs
lack the sensitivity of MS-based quantitation because they require more of a compound to be
present to provide MS/MS spectra of substantial quality. They also operate in data-dependent
acquisition (DDA) mode for switching from MS to MS/MS, which is not always consistent due
to sample and instrument variations, which will be discussed further in the following section.
Several MS and MS/MS-based tools have been developed in recent years to enable quantitation
of NPs without requiring prior knowledge of these compounds’ sequences. The ability to reliably
quantitate and identify NPs that change during an important biological process has been a great
boon to function-directed discovery of NPs.
3.3.2.
Data-Independent
MS/MS-Methods
for
Simultaneous
Identification
and
Quantification
3.3.2.1. The Need to Conduct Data-Independent MS/MS
Advances in the last few years have led to the development and implementation of new
methods for acquiring MS/MS data that allow true identification and quantification in a single
instrumental run by making acquisition of fragment ion spectra data-independent. These dataindependent acquisition (DIA) techniques include MSE, SWATH, etc. A recent review described
some aspects of these techniques in the context of phosphoproteomics [23]. As mentioned
previously, DDA MS/MS relies upon mass filtering of a single ion to be sent to the collision cell,
followed by MS analysis of its daughter ions, and switching from MS to MS/MS mode is
dependent on the ions observed in the MS1 spectrum. The addition of MS2 scans to the
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instrument’s duty cycle decreases the number of instrument scans that can be used for MS1
acquisition. With typical chromatography conditions, in which peak widths are on the scale of
minutes, most instruments then cannot obtain enough MS1 data points for proper peak integration
due to the allocation of some scans to MS2. In addition to not having enough data points per MS1
peak, the number of points per peak will vary in each sample due to data-dependent MS to
MS/MS switching, which is usually triggered by compounds reaching a threshold number of
MS1 counts. The time at which switching occurs and the raw number of ion counts can vary due
to slight variations in instrumental conditions over time. The concentration of the compound,
which is roughly proportional to the number of MS1 counts, will also vary in each sample. Thus,
instrumental and sample variability will influence how the instrument allocates scans to MS1 or
MS2, and thus lead to different numbers of MS1 points per peak in each run. For these reasons,
MS1 traces obtained via data-dependent acquisition (DDA) cannot be used reliably for area under
the curve (AUC) methods of quantitation, TMT methods may not obtain information about the
same precursors in different runs, and spectral counting will also experience variability.
Data-independent acquisition methods attempt to solve these problems associated with
DDA by producing fragment ions for all precursors equally, instead of selecting single highintensity parent ions. Prior to the advent of electrospray ionization (ESI), matrix-assisted laser
desorption ionization (MALDI), and related “soft” ionization techniques, “hard” ionization
methods including electron impact (EI) and chemical ionization (CI) were used as ion sources.
These techniques not only ionized analytes, but also forced them to degrade into many
components, and thus could be termed the first data-independent MS/MS instruments, as all
analytes experienced a degree of fragmentation upon ionization. These methods had limited
utility for biological samples (as opposed to small molecules) due to the complexity of the
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resultant spectra. This is a result of the increased numbers and masses of molecular species in
biological samples and the larger variety of functional groups where fragmentation can occur in
biomolecules. Highly efficient molecular separation was required prior to hard ionization in
order to provide interpretable spectra, and methods for separating hydrophilic biomolecules were
not as well developed or as compatible with ionization sources (which typically required analytes
to be in the gas phase) as were required.
3.3.2.2. Instrumental Techniques for Acquisition of Data-Independent MS/MS
Several of the earliest schemes for data-independent MS/MS of biomolecules relied upon
in-source fragmentation, a data-independent fragmentation method in which all analytes are
fragmented prior to entering the mass analyzer. One can alternate MS acquisitions or scans
between low fragmentation conditions and high fragmentation conditions to obtain scans of
parent and fragment ions, but this typically cannot be done on the time scale of a LC experiment
in order to provide quantitative information in addition to sequence identification. The first
experiment using alternating scans in a single LC run to identify peptides was reported by
Kosaka and colleagues in 2003 [24]. Infrared multiphoton dissociation (IRMPD) was applied on
alternating scans of Fourier transform ion cyclotron resonance (FTICR) MS of nanoLC eluent in
a method termed alternating-scan nano-LC/IRMPD-FTICR-MS. Two TICs were reconstructed
from these alternating scans, with one corresponding to normal FTICR (parents) and the other to
IRMPD-FTICR (fragments). Precursor masses from the parent scan were used to aid peptide
sequencing in the fragment scan. The authors used this method to analyze tryptic digests of BSA
and a protein purified from rat liver, with 55 and 46% coverage respectively [24]. Although the
authors did not conduct quantitation, the TIC traces obtained appear to be suitable for AUC
calculation. In-source fragmented bovine serum albumin (BSA) digests were also used by
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Masselon and co-workers in 2003 to demonstrate that MS/MS spectra of mixtures of peptides
could be interpreted without prior knowledge of which peaks corresponded to precursors and
which were fragments, provided accurate masses were obtained [25]. Although this work
demonstrated that it is not necessary to acquire spectra of parent and daughter ions separately,
this is a major component of most data-independent acquisition methods used in the intervening
years to reduce the complexity of MS/MS interpretation.
Alternating in-source fragmentation methods was first used for quantification in addition
to identification in 2003 [26, 27]. These techniques alternate source conditions between those
that produce high or low degrees of fragmentation to produce spectra containing primarily parent
or fragment ions, similar to the work by Kosaka et al. [24]. Purvine and colleagues termed this
“shotgun” CID, employed it for simultaneous ID and quantification, and suggested a method to
assign fragment ions to the appropriate parent ion using chromatographic retention profiles [27].
This same approach was also conducted in 2003 by Williams and colleagues, who also used a
sequence of multiple levels of in-source fragmentation for more complete peptide fragmentation
[26]. These methods were later incorporated into DIA techniques, i.e. alternating precursor and
fragment scans, using retention time profiles to assign daughter ions to parent ions, and multiple
levels of fragmentation efficiency (morphed into a collision energy ramp from low to high).
Another technique for DIA, using large precursor mass isolation windows and rapid
MS/MS scan acquisition, was proposed by Venable and colleagues in 2004 [28]. In this
technique, a rapidly scanning ion trap mass spectrometer was used. For a single MS/MS scan,
precursor molecules spanning a 10 Da window are accumulated in the ion trap, fragmented, and
analyzed. The entire m/z range of interest is divided into these 10 Da segments, which are
sequentially analyzed, and with the rapid scan speed of this type of MS instrument,
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approximately 40 of these scans can be conducted in 10 s, corresponding to a total range of about
400 Da, with larger mass ranges possible if lower quality scans or larger precursor windows are
used. Since the half-widths of most UPLC peaks are at least 30 s, this permits enough MS/MS
spectra to be obtained across the elution peak for quantitation in addition to the identification
information provided. Indeed, quantification was improved by increasing the signal-to-noise
ratio and the greater specificity that comes from quantification based on a daughter ion as
opposed to a parent ion. In addition to improved quantification concurrent with identification, the
number of identifications increased [28]. This technique of selecting subsequent “chunks” of the
mass range for fragmentation in a fast-scanning instrument is also currently used. These two
schema for data-independent analysis—no isolation of precursors, or isolation of “chunks” of
precursors in segments spanning the mass range of interest—underlie the mature forms of DIA
that have become commercially available in recent years.
A major hurdle in advancing DIA methods was that researchers outside of instrument
companies often did not have the ability to alter instrument firmware to employ these strategies.
Thus, further advancement was made primarily by researchers in instrument companies or in labs
with home-built instruments. Patent protection of these techniques has also limited the ability of
researchers outside of the companies that hold the patents to experiment with the techniques. The
two schemes of DIA initially developed, as well as some similar strategies, are thus now
proprietary technologies defined by the companies that hold the patents.
The idea of alternating between parent and daughter ion spectra by changing global
fragmentation efficiency, as put forth by the work of Kosaka and Purvine, was further advanced
by using different energies in the collision cell, instead of at the source, with a high-resolution,
high mass accuracy mass spectrometer by Waters Corporation. Beginning in 2005, Silva,
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Hughes, and co-workers advanced one version of this technique, termed MSE—as the data
acquired contains both normal and elevated Energy mass spectra—for protein/peptide analysis
[29-32]. The resulting daughter ions are then detected in spectra containing fragments from
multiple precursors (called chimeric spectra), and deconvolution is conducted to assign daughter
ions to parents, based on elution profiles and the knowledge that a fragment ion cannot have a
higher mass than the parent. This technique has been successfully used for simultaneous
identification and quantification of proteins in a number of studies, in addition to those already
mentioned [33-53], and is exclusive to instruments made by Waters. Another universal
fragmentation technique has been implemented with the Orbitrap (Thermo-Fisher) mass
spectrometer, called all-ion fragmentation (AIF). This technique sends alternating packets of ions
either to the detector directly, or to a collision cell before sending them to the detector [54].
Fragmentation of all precursors, a technique that grew out of alternating fragmentation efficiency
at the ion source, has been developed into two mature techniques for DIA, MSE on Waters
instruments and AIF on Thermo Orbitrap instruments.
The other main scheme for DIA MS—scanning large precursor ion windows as suggested
by Venable et al.—has also been developed commercially. Some non-commercial research was
initially conducted on this technique [55, 56], but it has subsequently been developed for use
with ABSCIEX TripleTOF instruments and termed sequential window acquisition of the total
high resolution mass spectrum (SWATH-MS) or MS/MSALL [57]. This technique is newer than
MSE, and studies using it are less common. A similar technique that employs smaller mass
ranges—2.5-3 Da isolated per MS/MS spectrum, isolation windows separated by 1.5-3 Da, 10-30
isolation windows monitored per run—and multiple sequential instrument runs to cover the
entire m/z range of interest has been developed, called precursor acquisition independent from
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ion count (PAcIFIC) [58-61]. This technique consumes large amounts of instrument time but
provides high quality identification and quantification data, and is platform-independent. A final
technique employs very large isolation windows (100 Da) on a Fourier transform ion trap mass
spectrometer, and is termed Fourier transform-all reaction monitoring (FT-ARM) [62]. For this
technique to work, the user must be able to set up the isolation windows for MS/MS acquisition
(i.e., the instrument firmware must allow this), and the software to interpret the data is opensource but currently only works with Thermo files. “Chunk” isolation of precursors has been
developed into SWATH-MS (MS/MSALL), PAcIFIC, and FT-ARM. Slight variations upon the
technique permit more independent research in this technique.
3.3.2.3. Data Analysis in Data-Independent MS
Chimeric spectra are the main challenge of DIA MS methods with both global and
sequential segmented precursor selection and fragmentation methods. New methods for data
analysis had to be developed to reduce the complexity of interpreting these spectra, as they have
challenges not typically encountered in DDA MS/MS spectrum identification: more than one
precursor mass is possible per spectrum, and the fragment ions present in a given spectrum can
correspond to one or more of these parent ions. In contrast, DDA spectra have an identified
precursor (the mass isolated in the first mass filter for fragmentation) and all fragments present in
the spectrum ideally correspond to that single precursor. A number of strategies have been
developed to interpret these spectra, and most are dependent on the method of acquisition of
spectra.
In 2003, Masselon and co-workers addressed the complexity of automated identification
of precursors for MS/MS spectra that contain fragments of multiple compounds, both when there
is limited precursor information (multiplexed MS/MS, a DDA method) or no precursor
142
information (true DIA, due here to in-source fragmentation)
[25]. The identification of
fragments produced by in-source decay was conducted by generating a list of possible peptides
for the species and enzyme specified. The b- and y- ions of these potential precursor peptides
were then calculated, and searched against the DIA MS/MS spectra obtained. Using this method,
peptides from a BSA digest were identified by searching databases from the E. coli and C.
elegans genomes, with the sequence of BSA added to them. Many identifications were made for
BSA peptides (138 true positive peptide matches), with only a small number of false positives—
3 (2.8%) with the E. coli database, and 11 (7.8%) with the C. elegans database. This work
bridges the gap between multiplexed DDA MS, for which interpretation of chimeric spectra was
difficult but possible, and DIA MS, which has less information than multiplex MS/MS because a
discrete set of possible precursor molecules is not known.
Interpretation of chimeric spectra typically requires them to be “deconvoluted,” a process
that describes the assignment of the mixed fragment ions to the appropriate precursors. This is
normally achieved by using high resolution MS data with optimal separation. Ideal separation
will allow every molecular entity in the sample to be distinguished by their analytical separation
efficiency, and daughter ions can thus be assigned to parent ion. This degree of separation, in
which all distinct compounds have different separation profiles, is not easy to achieve. The
addition of another dimension of separation to the traditional UPLC-MS/MS experiment has
been beneficial in this regard. Several studies have conducted multistage LC prior to ionization
[36, 48].
MSE is marketed in some instruments that also have ion mobility separation
capabilities, and this additional gas phase separation provides better resolution of analytes.
Retention time (or drift time, in the case of ion mobility) can aid in deconvolution of chimeric
DIA spectra.
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Other information can be used to aid in deconvolution of chimeric spectra. In the cases of
mass spectra acquired from sequential segments of the m/z range selected independently (as in
SWATH-MS and related techniques), limited information about the precursor is available,
namely that its m/z falls within the segment isolated at that moment. General principles of
fragmentation can also be used for deconvolution, such as the fact that daughter ions will not
have a greater monoisotopic mass than their parents. Instrument-specific software is typically
used to make DIA MS data more easily interpreted by software originally developed for DDA
data, but open-source software is becoming more common for deconvolution of chimeric spectra,
including FT-ARM [62] and DeMux [55].
3.4. Novel Sampling Methods for MS-Based Functional Discovery of Neuropeptides
In addition to new MS tools for quantification of NPs, different sampling methods for use
with MS-based analysis have also been established. These techniques can provide more
significant temporal information about changes in NP content, or can be used to determine the
localization of NPs within well-defined neural organs or neural circuits that have areas with
known function.
3.4.1. Live-Animal Sampling for Correlation of Neuropeptide Changes with Behavior
Many MS-based functional discovery studies rely on the use of tissue-based sampling
techniques which typically require sacrificing multiple animals for each biological sample, such
as brain or ganglion homogenates. Although these sampling methods have been used to assign
function to many NPs, high degrees of inter-animal variability in baseline levels of NPs can
obfuscate net changes that may be important to NP function. Obtaining multiple samples from a
single animal is thus highly desirable. Bodily fluids may be sampled via needle, but in mammals,
the blood-brain-barrier (BBB) prevents changes in many NPs from being reflected in circulating
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levels of these compounds, and obtaining cerebrospinal fluid (CSF) is invasive. For crustaceans,
the BBB is less complex, and sampling from the hemolymph provides greater information about
active NPs [63]. However, hemolymph and CSF contain a wide variety of other components, and
obtaining a sample of hemolymph from a marine animal via conventional syringe withdrawal
technique induces a great deal of stress to the animal.
The optimal sampling method would allow multiple samples to be taken from the animal
on a time course relevant to the experimental manipulation without disturbing the animal. Thus,
liquid-based sampling is preferred, and dynamic sampling on-line with the animal (i.e.
microdialysis (MD), introduced previously) would be the best. On-line sampling allows
continual collection of sample without disturbing the animal’s behavior. Two main methods for
this type of sampling exist: microdialysis and push-pull perfusion. Cannulation can also be
conducted but can lead to animal perturbation due to removing liquid from the animal without
replacing the volume and salts lost. Push-pull perfusion is a variation on cannulation that
employs an inlet cannula in addition to the withdrawal cannula in order to replace the liquid
removed with an equal volume of physiological fluid. To the authors’ knowledge, the only online sampling technique successfully implemented in crustaceans is microdialysis, likely due to
the difficulty of surgery on the crab. In the authors’ experience and previously published work,
crabs do not heal well from insult to the carapace as opposed to the soft tissue at joints due to
limited glue adhesion to the shell. However, this technique has been used successfully to sample
from crayfish [64] and the crabs Cancer borealis [65, 66], Callinectes sapidus, Carcinus
maenas, and Cancer irroratus.
MD has long been used in neuroscience to sample NPs and neurotransmitters directly
from the brains of mammals, but its use in conjunction with MS-based analysis approach has
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only recently been established (reviewed in [67, 68]). MD employs a probe that is surgically
implanted into the tissue of interest. At the tip of this probe is a dialysis membrane, through
which molecules from the area outside the probe can diffuse into the probe. Liquid is perfused
through the probe at a low flow rate (µL/min), and is collected at the probe outlet. This liquid
carries with it molecules that diffuse through the dialysis membrane into the probe according to
their concentration gradient. After recovery from the surgical implantation, animals can behave
normally, and thus with MD, molecules can be collected from an animal while it is behaving
normally with minimal perturbation. This allows maximal correlation of behavior with
neurochemistry, and permits multiple samples to be taken from the same animal, to limit the
impact of inter-animal variability. The pores of the dialysis membrane also only permit
molecules below a certain molecular weight (the molecular weight cutoff (MWCO)) to pass into
the probe, thus reducing sample complexity, an advantage not obtained with push-pull perfusion.
Additional work needs to be conducted, however, to improve the sensitivity of analysis
for microdialysates, as the temporal resolution currently available with the technique is 4 hours
or greater. This is due primarily to the low concentration of NPs present in the hemolymph, as
well as the dialysis membrane impeding the influx of molecules with weights below the MWCO
but greater than a few hundred Da. The percent transfer of molecules through the membrane
decreases in an exponential manner as mass increases, with the MWCO typically representing
the mass at which 95% of molecules will not pass through. Different probe membrane materials
may also impact the recovery of larger molecules. Affinity agents can be added to the liquid
perfusing the probe to increase the net flux of analyte into the probe. These affinity agents,
usually solids with some molecule with NP affinity attached to their surface, remove the bound
analyte from the diffusion equation that controls flow of the analyte inside at the probe tip. This
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method, termed affinity-enhanced microdialysis (AE-MD), was pioneered by Stenken and
colleagues in vitro and in mammalian systems [69-81]. Application of AE-MD in crabs has
increased temporal resolution to 30 minutes, and the concentration change of neuropeptides
following feeding can be observed with this technique.
On-line sampling from invertebrates is not simple, particularly for smaller invertebrates,
due to the challenge of implanting a MD probe or cannulae into an animal with an exoskeleton. It
also suffers from reduced sensitivity due to the low concentration of NPs in the extracellular
fluid. However, the benefits of sampling from the animal without disturbing it and the ability to
obtain multiple time points from the same animal make it a very attractive option for generating
functionally relevant information about NPs.
3.4.1.1. Methods of Analysis for Microdialysates
Typical methods for quantitative analysis of microdialysates (the liquid collected at the
probe outlet) include immunochemistry and chromatographic separations coupled with
electrochemical, ultraviolet-visible spectroscopy, photometric, and/or SRM/MRM-based MS
detection methods (reviewed in [7, 67]), all of which require a priori knowledge of the NP’s
structure or chromatographic properties. A number of NP MD studies using immunochemical
detection have been conducted, however, these analyses often suffer from antibody crossreactivity with the NP of interest. As a result, it is often difficult to clearly assign functions to
single molecular entities, and there are instances in which NP isoforms with slight variations in
sequence have different biological effects. Although these quantitative analyses of MD samples
are often part of studies of NP function, they usually rely upon quantitation that is less specific
(antibody-based) and/or require prior knowledge of the NP’s structure (targeted MS techniques
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such as SRM and AQUA), and thus are less well suited for discovery of NPs based on global
comparative analysis of NPs from different functional states.
Studies where MS-based techniques are used for discovery of NPs found in dialysate, in
contrast, usually do not also include quantitative analysis, and thus provide only minimal
functional information [65, 66, 82, 83]. A limitation of MS-based functional discovery for NPs is
that it is difficult to obtain sequence information about novel NPs concurrent with quantitative
analysis methods described previously, except when TMTs or spectral counting are used. Most
tandem MS instruments are operated in a mode in which they dynamically switch between MS
and MS/MS. Sequence information is available only from MS/MS scans, which are added to the
duty cycle of the instrument in place of MS scans. This addition to the duty cycle leads to less
frequent acquisition of MS data. Therefore, the number of MS data points obtained per peak
decreases during the run in a data-dependent manner, and this is not reproducible between runs.
Integrating the AUC for the ion of interest is not reproducible for data acquired when dynamic
MS to MS/MS switching is used. Separate instrument runs must be conducted for identification
based on MS/MS sequence and quantification based on MS, for which ions can be identified by
their m/z and chromatographic properties.
3.4.1.2. Quantitation of NPs in MD Samples with Targeted MS Methods for Elucidation of
Function
MS quantitation methods that require prior knowledge of NP sequence to quantitate these
compounds in dialysates from mammals have been reported previously [84-88]. MD studies in
invertebrates are less common. Several related papers that demonstrate such a targeted approach
to MS-based quantitation of NPs in MD samples will be highlighted here to demonstrate the
potential of MD used in conjunction with MS. Although they are not function-driven
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experiments, the same sampling techniques could be used with non-targeted MS approaches to
conduct function-driven NP discovery. In these studies, capillary LC (capLC)-MS/MS [89] and
capLC-MS3 [90-92] using SRM of known fragmentation reactions for selected NPs was
conducted to quantitate NPs collected via MD from the basal ganglia of anesthetized and freelymoving rats under baseline and stimulated conditions. As mentioned previously, SRM requires
that the structure of the analyte and its daughter ions is known prior to analysis. MS3 for SRM
adds another level of MS analysis, in which the daughter ions generated upon MS/MS analysis
are selected and fragmented, and a specific grand-daughter ion of this 2nd order fragmentation is
monitored. These targeted MS techniques provide stronger evidence for a correlation between
NP concentration changes and stimulation, but require a priori knowledge of the NPs in question
and thus cannot be used in a function-driven discovery experiment.
These studies used two types of stimulation, which provide different amounts of
information about the function of the NPs monitored. Several employed K+-induced widespread
neuronal depolarization in the globus pallidus (GP) [89] and striatum [90, 91] to induce the
release of enkephalins [89-91] and dynorphins [91]. Although the non-specific nature of this
stimulation does not permit assignment of an exact functional role for these NPs, correlating
their release more generally with activity in the basal ganglia may point toward a functional role
related to the overall functions of these subnuclei.
The most recent of these studies also employed capLC-MS3 quantitation but used more
biologically relevant manipulations concurrent with behavioral monitoring to determine a more
specific function for these compounds [92]. This study quantitated enkephalins in GP
microdialysate from rats along with locomotor behavioral analysis, monitoring neurotransmitter
(NT) concentration changes, and pharmacological manipulations. The SRM capLC-MS3
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quantitation system mentioned above was used to quantitate [Met]- and [Leu]-enkephalin (ME
and LE) concurrent with administration of drugs that increase dopamine levels (amphetamine) or
are dopamine receptor 1 or 2 (D1R or D2R) blockers. The researchers were able to determine that
ME and LE increase after administration of amphetamine, and that this effect primarily requires
the D1R. Further monitoring of NT concentrations and locomotor behavior during administration
of D1R, D2R, and opioid receptor antagonists permitted the development of a model for the role
of ME and LE in locomotor function mediated by the GP. This study is highly significant due to
the great specificity with which the authors were able to determine excitatory and inhibitory
effects of neurochemicals. This was accomplished through the combined use of MS-based
quantitation of NPs, a novel method for NT quantitation, behavioral modeling, and selective
pharmacologic agents. This targeted study determined a specific function for ME and LE, based
on MS analysis of MD samples.
3.4.1.3. Discovery of NPs in MD with MS/MS Tools
MS tools can also be used to identify unknown NPs, and a number of such studies have
been conducted [82, 93-95]. One such work is complementary to the ME and LE quantitation
studies, and consists of MS/MS-based discovery of NPs from the striata of rats under K+stimulated conditions [83]. This study identified 29 peptides from 6 proteins, 25 of which were
isoforms that had not previously been observed. The combination of NP identification with the
quantitation studies discussed previously comprises an alternative approach to function-driven
NP discovery. The decision of which NPs to quantify was based on previously known
information about these NPs. A function-driven discovery experiment would instead look for
compounds with interesting changes throughout the course of an experimental manipulation, and
then attempt to identify them [65]. The power of functional discovery of NPs via MS-based
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quantitation methods discussed in this article is that the data obtained upon experimental
manipulation itself determines which NPs are quantified and subjected to further study.
3.4.1.4. Experimental Design for Functional Discovery of NPs in MD Samples
True function-driven discovery of NPs requires functional relevance to guide the choice
of which compounds to study further. This can be achieved in a number of ways, but has rarely
been done with MD. MS-based relative quantitation of several isoforms of dynorphin A (Dyn A)
was conducted on samples collected from rats with a unilateral lesion of the striatum [96]. The
identity of the isoforms was then determined by peak mass matching and MS/MS analysis. This
study revealed qualitative and quantitative differences in the processing of Dyn A in the lesioned
striatum. By quantitating all peaks of interest and later determining their identity, this study
conducted function-driven discovery of NPs.
MS/MS-based quantitation techniques for function-driven discovery have been used
rarely with MD samples. These techniques permit quantitation and identification in a single run,
without prior information, but they often require larger amounts of sample, which makes them
unpopular for MD. A study of this type used spectral counting to characterize the NP content of
the striatum of the rat brain under baseline and K+-stimulated conditions [97], similar to the
studies mentioned previously. In this work, 88 proteins and 100 endogenous peptides from 26
protein precursors were identified and quantified. The relative abundances of these compounds
in dialysate based on spectral counts were determined, and these relative abundances were
observed to change upon K+-depolarization. NPs made up a greater proportion of the
components observed in microdialysate after depolarization. In addition, the relative abundances
of two NPs, proenkephalin A precursor and a VGF-related peptide, were greater postdepolarization. As the method of stimulation and location of probe placement was similar to
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previously mentioned studies, the amount and type of functional information obtained from this
study is also similar, although additional compounds not previously detected were found and
their quantity changes determined. One caveat is that a number of large proteins were detected,
including many above the MWCO of probes used. This suggests possible damage to the tissue
surrounding the probe causing large-scale protein degradation. However, this study acquired
both structural and quantitative information in the same LC-MS/MS run, which is a great boon
for function-driven discovery of NPs.
MD has been conducted on alert Jonah crabs (Cancer borealis) under baseline conditions
with MS and MS/MS analysis of the resulting samples [65, 66]. Although no quantitative results
were available in that study, 35 NPs previously identified in C. borealis were detected. A major
limitation in the application of this scheme for function-related discovery of NPs was the long
collection time of MD samples. Collections were at minimum 4 hours, which is not compatible
with studying most of the dynamic processes thought to be mediated by NPs. This long
collection time was required due to the low sensitivity of the systems used for detection. New
advances in UPLC-MS and improvements to the MD collection technique termed affinityenhanced microdialysis have made it possible to follow concentration changes in NPs on a
shorter time scale with 30 min sampling intervals [98]. These enhancements have made it
possible to determine the concentration changes in NPs induced by feeding in C. borealis, and
have provided additional evidence that several NPs have feeding-related functions. Further
refinement of the techniques used may allow for collection of samples on an even faster time
scale, which will provide useful information about the time-resolved changes in these
compounds with respect to feeding, and thus aid in elucidating the NPs involved in this
important physiological event. MD sampling of alert, behaving animals, combined with MS and
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MS/MS techniques described previously, allows for tight correlation of NP changes with
behavior, and thus is a useful technique.
3.4.2. Mass Spectrometry Imaging of Tissue to Correlate Neuropeptide Location with
Function
Not only do MS-based tools permit NP function to be elucidated by correlating behavior
with release over time, they allow determination of NP localization within tissue as a guide for
function. Mass spectrometry imaging (MSI) is an emerging technique, in which a tissue of
interest is sectioned and analyzed with MS (recent reviews [4, 99-104]. A coordinate system is
superimposed on the tissue, and mass spectra are acquired for and registered to positions within
this system. The intensity of a given peak in the spectra can be compiled into a heat map image
to represent the distribution of that compound throughout the tissue. This technique has been
applied with great success to determine the localization of NPs within the brains of several
species (see selected reviews and relevant invertebrate papers [3, 99-115]). Previous knowledge
about the function of certain brain structures can then be extrapolated to NPs that are heavily
concentrated in these structures.
The brain of the decapod crustacean has been studied extensively, and several regions
have known functions due to the locations from which they receive inputs and to which they
send outputs. For instance, the olfactory neuropil (ON) and antennal lobes (AL) in the brains of
decapod crustaceans both receive input from olfactory receptors, although the AL receives
higher-order olfactory input—the olfactory signals are relayed through a set of interneurons
before they reach the AL—and input from other senses [116]. In an IMS study of the brain of H.
americanus, a number of patterns of NP expression were determined, including a greater
expression of two SIFamide family peptides in the ON, and increased levels of two orcokinins in
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the AL [113] (shown in Fig. 3.4). The difference in localization of these two families of peptides
may indicate different functional roles, i.e. SIFamides are more important in primary processing
of olfactory signals, and orcokinins are involved in higher-order processing, including
integration of signals from multiple sensory modalities. MSI is a useful tool that allows
assignment of function to a NP based on its localization patterns within nervous structures with
known functional areas. In conjunction with novel tools for quantitation with MS, advances in
sampling have allowed the identification and assignment of NP function by providing
information about their temporal and spatial distribution.
3.5. Validation of Potential Function for NPs Discovered via MS-Based Methods
Although function-driven discovery of neuropeptides using MS-based tools provides
definitive information about NP identity, the functional information is often less complete; more
targeted studies are frequently required for definitive assignment of function. Putative functions
identified by MS-based functional discovery of NPs can be further investigated using a variety of
targeted tools, including more traditional bioassays, “omics”-based approaches, and some
previously mentioned MS techniques. Similarly, many of the methods used for functional
discovery of NPs can also be used to determine the identities of compounds with functions
already known. Thus, a cycle of identifying compounds and determining their functions forms
the basis of neuropeptide research (Fig. 3.1). Some of the techniques commonly used to validate
the putative functions of NPs discovered by non-targeted MS strategies will be described.
There are many methods to determine the role a NP plays in a biological system. NPs can
be administered to an animal, and neurochemical and/or behavioral changes can be monitored.
Endogenous NP content also can be monitored during the course of a physiological change to
provide correlations between NPs and functions. Although the first technique provides a more
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definitive causal relationship, it requires the administration of a pure sample of a known
compound, and a well-defined behavior or physiochemical change to be monitored. Thus, a great
deal of foreknowledge is required to carry out this type of functional relationship study, and it is
best suited to verification of postulated functions. The second type permits discovery of
previously unknown NP-function relationships but provides less definitive information.
3.5.1. Causal Studies of NP Function
Two main methods have been used to monitor NP action in decapods via direct
administration. These are ex vivo nervous system or chromatophore preparations, monitored by
electrochemical recording or microscopy, and in vivo injection of the decapod with the
substance, followed by monitoring behavioral and/or hemolymph chemical changes. The ex vivo
preparation allows—via clever use of pharmacological agents and electrophysiological
techniques—determination of the mechanism of NP action at the molecular level. An in vivo
experiment captures whole-animal behavioral changes that do not translate well to the cellular
level and changes that require the involvement of multiple organ systems, such as metabolism of
stored energy.
3.5.1.1. Ex Vivo NP Function Experiments
A common method for determining the function of a putative NP is to isolate a neural
organ, muscle, or portion of the nervous system, and apply the NP to the preparation. Changes in
the electrical profiles of neurons and muscles or in the contraction frequency or strength of a
muscle can then be recorded, and thus are correlated with the NP applied. These studies, in
which an organ or muscle is removed but kept alive and used as a test bed for NP action, can be
termed ex vivo studies. A number of different ex vivo preparations have been described in
invertebrates, but the classic model is the isolated crustacean stomatogastric nervous system
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(STNS), a set of ganglia connected by nerves located near the stomach. For detailed instructions
on this preparation, see videos and articles by Gutierrez and Grashow and Tobin and Bierman
[117, 118]. Fig. 3.4 illustrates the basic preparation and how the function of a bath-applied NP
can be determined using this system. The STNS comprises a central-pattern generating circuit in
control of feeding, with each cell in each ganglion having a characteristic rhythmic firing pattern.
The cell number, location, and connectivity in the STNS are nearly identical in all mature
members of each species. For causal studies of NP function using this system, the STNS is
dissected and placed in an electrophysiological recording dish, and a variety of electrodes are
placed in selected cells and on nerves. NPs can then be applied to certain cells, ganglia, or the
entire preparation, and alterations in the firing frequency and pattern of these cells can be
recorded. Two distinct patterns are observed, one to control chewing—the gastric mill rhythm—
and one to control filtering of food—the pyloric rhythm. The addition of pharmacological agents
with known function along with the NPs or modern electrophysiological techniques such as
voltage clamping can allow for elucidation of their exact molecular targets. This system was
pioneered by Maynard and extensively used by Marder and colleagues to determine the function
of several NPs on the rhythmic patterns of the gastric mill and pylorus. For a review of the
known functions of NPs on this system, see a review by Marder and Bucher [119]. The ex vivo
STNS preparation allows for identification of the molecular mechanisms by which a NP acts on
the cells in this system, and has provided important information on the function of many
crustacean NPs.
Isolated chromatophores (cells in the epithelium or eye that change color) have been used
to determine the activity of several pigment-related hormones. Simply, chromatophores are
isolated from the eyestalk, placed on a microscope slide, and NPs are applied [120, 121]. This
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causes pigments in the cell to change their distribution, which can be observed via microscopy.
The molecular signaling pathways by which externally applied NPs cause redistribution of
pigments in a crustacean chromatophore are varied depending on whether the NP is pigmentdispersing or pigment-concentrating. For further information, see the sections on pigmentaffecting hormones in Chapter 2. The ex vivo STNS and chromatophore preparations can be used
to determine the molecular targets and functions of certain NPs that act on cells in these systems.
This is useful for NPs that act very specifically there, but less useful if the action of a NP is
distributed across multiple organs.
3.5.1.2. In Vivo Administration of NPs to Determine Function
NPs can also be administered to the animal as a whole, and changes in behavior or
bioactive chemicals can then be monitored. Provided administration of the NP causes a change
greater than that seen following administration of a carrier solution (physiological saline), that
NP can be assumed to have exacted that change. Administration is typically conducted via
injection of a solution into the soft tissue near a joint, although implantation of a permanent
cannula would also be appropriate if multiple doses were required. Behavioral changes
monitored might include the rapidity of movement, frequency of a certain pose being assumed,
or movement of certain parts of the body, such as the tail or mouth parts. Biochemical changes
caused by NP administration can be determined by sampling the extracellular fluid/blood of the
crustacean, again through the soft tissue at a leg joint. Although microdialysis is appropriate for
both NP administration and sampling from the hemolymph in this type of experiment, and is
indeed preferable in terms of invasiveness, it has not yet been applied for either of these roles in
the crustacean.
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An NP can also be administered in vivo and hemolymph chemical changes or behavioral
changes can be monitored. A recent study of this type has been presented by Chung et al. [122],
in which crustacean hyperglycemic hormone (CHH) was administered to eyestalk-ablated
Chionoecetes bairdi, after which hemolymph glucose levels were monitored.The eyestalk is the
primary endogenous source of CHH in the crustacean, and removing it provides better causal
evidence that the CHH injected led to the increases in hemolymph glucose level observed. By
this method, Chung and colleagues were able to demonstrate CHH’s role in the mobilization of
stored energy, a process that requires the NP to act on multiple organs. Both ex vivo and in vivo
studies can be used to determine the causal relationship between NPs and functions, on a protein,
cell, or organismal level. However, these causal relationship studies require administration of a
pure NP sample and monitoring of a well-defined output that is already postulated to be the
function of the NP. In addition, they typically only allow for determination of the function of a
single NP at a time, and it is well known that NPs can attenuate and alter each other’s function.
3.5.2. Correlational Studies of NP Function in Invertebrates
Due to the drawbacks of causal studies of NP function in invertebrates mentioned above,
correlational studies are often more suitable. When mass spectrometry is used, many methods for
causal study of NP function are identical to the function-driven discovery methods discussed
previously. Most of previously mentioned MS tools that improve quantitation of multiple NPs
across different time points can be used when the chemical identity of the NP is known, often
with better success than when it is unknown. There is an inherent disadvantage to correlational
studies in that they do not have the specificity in determining NP-function relationships or
functional unit resolution that is seen in causal experiments, but they are an important part of
determining the putative function of a NP.
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For correlational studies, the neuropeptide content in several time points or different
conditions must be determined. A number of challenges arise with this requirement; namely what
type of sample to obtain (liquid or tissue) and how to conduct quantitation. The previously
discussed quantitation schemes and novel sampling methods are of particular use for this
component of causal studies of NP function.
3.5.3. Peptidomics to Genomics: Function Determination by Homology Search of Public
Databases
MS-based discovery of NPs can also be combined with homology searches to verify
function. The function of many NPs is conserved throughout evolution [1, 123-128]. Recent
reviews have discussed the technique of database mining for discovery of novel NPs [8, 129131], and searching sequences obtained via MS for homologous peptides in other related species
follows a similar procedure. A novel NP sequence identified by MS/MS can be searched against
the publicly available translated nucleotide database of expressed sequence tags (ESTs) using
tblastn. The EST database contains a large number of sequences from many organisms and is
constantly being expanded. Protein BLAST is not suitable for most peptides due to their short
sequences. Searches of this type may yield a homologous peptide in a different species, for
which the function is already known. Due to the conservation of function for many NPs across
disparate species, a putative function for a NP can thus be verified. Many NPs have been
predicted from bioinformatics analysis of publicly accessible databases, verified to exist in vivo
via immunochemical or MS methods, and/or determined to have biological function via
bioassays (notable invertebrate examples [127, 130-145]). Homology searching is not only a
tool for identification of NPs, but also provides information to their function and can verify the
results of a function-driven discovery study.
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3.6. Summary and Perspective
New tools and approaches that rely upon mass spectrometry and novel sampling methods
have been developed that allow researchers to pursue non-targeted function-driven discovery of
new compounds. These tools have the unique advantages of allowing functional information
about NPs, whether it comes from following concentration changes over time or determining the
distribution in a tissue, to be acquired concurrent with or before determination of the sequences
of these compounds. The power of this approach is that all NPs with relevance to a particular
function can be analyzed; one does not have to determine which compounds will be the focus of
an experiment prior to conducting it. The added benefit of MS’s ability to distinguish between
related NP isoforms increases the power of these analyses and permits knowledge of the exact
molecular entity that is active in the biological system. These powerful tools also enable
deciphering functional consequences of NP multiplicity and structural diversity.
In fact,
previously unknown and unpredicted isoforms of mammalian NPs have been observed in MSbased functional discovery studies via the analysis of microdialysate [89, 97]. Previously, a
molecular entity had to be characterized prior to determination of its function; now MS-based
function-driven discovery can identify NPs with putative functions that can then be validated
with more conventional means. In this way, NP identification and functional determination can
be accomplished in a cycle (Fig. 3.1), with multiple iterations providing a more complete picture
of both the NPs’ identities and functions. By eliminating the need for prior knowledge, new and
exciting NPs may be discovered with important functional roles, and vice versa.
In addition, the field of mass spectrometry has experienced constant growth and
innovation, and advancements in MS technology improve the sensitivity, speed, dynamic range,
and accuracy of analysis on a regular basis. Although the NPs observed most in current MS
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studies are likely to be the most abundant in vivo, future improvements will lead to elucidation of
roles for those NPs that are currently below the limits of detection or quantitation for current
instruments. By employing MS in function-driven non-targeted NP discovery workflows, even
greater amount of information will be able to be achieved as new technology is developed. The
types of experiments that will be possible with future generations of MS instrumentation will
allow greater information to be gained about the molecules that mediate intrinsic physiological
properties.
3.7. Works Cited
1. Li, L. J.; Sweedler, J. V. Peptides in the Brain: Mass Spectrometry-Based Measurement
Approaches and Challenges. Annu Rev Anal Chem. 2008, 1, 451-483.
2. Morimoto, R.; Satoh, F.; Murakami, O.; Totsune, K.; Saruta, M.; Suzuki, T.; Sasano, H.; Ito,
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142. Montagne, N.; Desdevises, Y.; Soyez, D.; Toullec, J. Y. Molecular evolution of the
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146. Schmerberg, C. M.; Li, L. Function-Driven Discovery of Neuropeptides with Mass
Spectrometry-Based Tools. Protein Pept Lett. 2012.
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3.8. Figures
Figure 3.1. Schematic representing the different types of MS-based tools that can be used for
function-driven NP discovery, by providing quantitative information about different samples. In
this schematic, samples are taken from animals either before and after an experimental
manipulation, or several times during the manipulation. Each sample is characterized with a
distinct color. MS and MS/MS-based techniques, both those that incorporate isotopic labels and
those that do not, are illustrated in the chart below. Further details of these techniques and
explanations of abbreviations are found in the text. From [146] Copyright 2012 Bentham Science
Publishers.
171
Figure 3.2. The role of function-driven NP discovery in the iterative cycle of neuropeptide
identity and function determination. A number of techniques exist to determine NP function that
require prior identification of the compounds, which was previously done with methods devoid
of input from functional studies. Function-driven MS discovery permits discovery of
functionally-relevant NPs via comparative peptidomic analysis. The putative functions thus
identified can be validated with other tools. Functions previously determined with less specific
analytical tools can also be verified and the exact identities of functionally-relevant compounds
determined. This permits cyclical study of NPs to better understand their identities and functions.
From [146] Copyright 2012 Bentham Science Publishers.
172
Figure 3.3. A) Workflow for differential display (DD) of NPs with expression changed after
feeding in C. borealis. B) Representative MALDI-TOF/TOF mass spectrum of labeled mixture
of brain extracts from fed and unfed crabs. The heavy labeled peaks (fed crabs, n=3) are
indicated with open circles, and the light labeled peaks are indicated with closed circles (unfed
crabs, n=3). Several abundant neuropeptides are indicated and labeled with their corresponding
amino acid sequences. Adapted with permission from Chen, R., Hui, L., Cape, S. S., Wang, J,
and Li, L. ACS Chem. Neurosci. 2010, 1 (3), pp 2-4-214. Copyright 2009 American Chemical
Society. (Ref. [10]) From [146] Copyright 2012 Bentham Science Publishers.
173
Figure 3.4. I) schematic illustrating MALDI imaging workflow. Adapted with permission from
Chen, R., Hui, L., Cape, S. S., Wang, J, and Li, L. ACS Chem. Neurosci. 2010, 1 (3), pp 2-4-214.
Copyright 2009 American Chemical Society (Ref. [10]). II) MALDI imaging of neuropeptide
localization in the H. americanus brain using both MALDI TOF/TOF and MALDI LTQ Orbitrap
mass spectrometers. (a) Schematic drawing of the lobster brain, which contains multiple
neuropils, including anterior (AMPN) and posterior medial protocerebral neuropils (PMPN),
olfactory lobe (ON), accessory lobe (AL), antenna I neuropil (AnN) and lateral II antenna
neuropil (LAN). Ion images of (b) VYRKPPFNGSIFamide (m/z 1423.8) and (c)
NFDEIDRSGFGFN (m/z 1517.7) were obtained using MALDI TOF/TOF. Ion images of
multiple known neuropeptides were acquired by MALDI LTQ Orbitrap, including: (d)
VYRKPPFNGSIFamide
(m/z
1423.8);
(e)
APSGFLGMRamide
(m/z
934.5);
(f)
NFDEIDRSGFGFN (m/z 1517.7); and (g) overlay of the above three neuropeptides. A novel
peptide HI/LASLYKPR (m/z 1084.6) in the lobster brain was also mapped using MALDI LTQ
Orbitrap instrument by (h) precursor ion scanning of m/z 1084.6 and (i) selected reaction
monitoring of transition between m/z 1084.6 and sequence-specific fragment ion m/z 685.4 (b6).
Adapted with permission from Chen, R., Jiang, X., Conaway, M. C. P., Mohtashemi, I., Hui, L,
Viner, R., and Li, L. J. Proteome Res.. 2010, 9 (2), pp 818-832. Copyright 2009 American
Chemical Society (Ref. [113]). From [146] Copyright 2012 Bentham Science Publishers.
174
175
Chapter 4. Mass Spectrometric Detection of Neuropeptides Using Affinity-Enhanced
Microdialysis with Antibody-Coated Magnetic Nanoparticles
Adapted from Schmerberg, C. M. and Li, L. Anal. Chem. [resubmitted]
4.1. Abstract
Microdialysis (MD) is a useful sampling tool for many applications due to its ability to
permit sampling from an animal concurrent with normal activity. MD is of particular importance
in the field of neuroscience, in which it is used to sample neurotransmitters (NTs) while the
animal is behaving in order to correlate dynamic changes in NTs with behavior. One important
class of signaling molecules, the neuropeptides (NPs), however, presented significant challenges
when studied with MD, due to the low relative recovery (RR) of NPs by this technique. Affinityenhanced microdialysis (AE-MD) has previously been used to improve recovery of NPs and
similar molecules. For AE-MD, an affinity agent (AA), such as an antibody-coated particle or
free antibody, is added to the liquid perfusing the MD probe. This AA provides an additional
mass transport driving force for analyte to pass through the dialysis membrane, and thus
increases the RR. In this work, a variety of AAs have been investigated for AE-MD of NPs in
vitro and in vivo, including particles with C18 surface functionality and antibody-coated
particles. Antibody-coated magnetic nanoparticles (AbMnP) provided the best RR enhancement
in vitro, with statistically significant (p<0.05) enhancements for 4 out of 6 NP standards tested,
and RR increases up to 41-fold. These particles were then used for in vivo MD in the Jonah crab,
Cancer borealis, during a feeding study, with mass spectrometric (MS) detection. 31 NPs were
detected in a 30 min sample, compared to 17 when no AA was used. The use of AbMnP also
increased the temporal resolution from 4-18 hrs in previous studies to just 30 min in this study.
The levels of NPs detected were also sufficient for reliable quantitation with the MS system in
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use, permitting quantitative analysis of the concentration changes for 7 identified NPs on a 30
min time course during feeding.
4.2. Introduction
Microdialysis (MD) is a sampling technique that allows collection of signaling molecules
from an animal while it is alert and behaving, with minimal disturbance to the animal. In this
technique, a MD probe is implanted into the tissue of interest and perfused with liquid at a flow
rate in the range of 0.1-10 µL/min. The tip of this MD probe consists of a dialysis membrane,
having pores with a defined molecular weight cutoff (MWCO). Molecules below this MWCO
near the tip of the probe passively diffuse into the probe and are then carried by the slowlymoving liquid out of the probe, through a length of tubing, and finally to a sample collection vial
or analysis system. This technique has been used successfully to collect a variety of different
molecules from a number of tissues in several species, and has provided important insights into
the action of compounds in vivo in a minimally perturbed animal [1, 2].
MD is of great utility in neuroscience, in which time-resolved changes in neurochemistry
during the performance of a behavior or exposure to a stimulus are of interest. Continual
collection of neurochemicals without disturbing the animal to obtain the samples allows the
experimenter to determine the molecular underpinnings of neuronal activity related to these
events, in the absence of any sampling-induced neuronal changes. MD has been used
successfully to monitor small molecule neurotransmitter (NT) changes in vertebrate animals
under a variety of different conditions, and has contributed greatly to our understanding of the
effects of NT release on behavior [1, 3-5].
One area that is particularly challenging for MD sampling is the analysis of larger
molecules, such as neuropeptides (NPs), which are below the MWCO of the probe but are in the
mass range of 500-10,000 [5-9]. A number of complex factors make recovery of NPs difficult.
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One such reason is the lower relative recovery (RR) of NPs in comparison to small molecules
due to their larger size hindering passage through the dialysis membrane. The RR is calculated
by taking the concentration of an analyte collected through MD divided by the concentration
outside the probe, and is usually expressed as a percentage. This RR is inversely related to the
mass of the molecule in a nonlinear manner, with larger molecules typically having RRs of less
than 50%. Another challenge is the low endogenous concentration of these compounds. NPs are
present in vivo at the nM - pM concentration range [2]. Therefore, the concentration collected,
governed by the laws of passive diffusion, is reduced compared to NTs due not only to their low
endogenous concentrations but also their reduced RR. Furthermore, RR is also governed by the
amount of time the liquid is in contact with the membrane (the MD flow rate, FR), with lower
flow rates leading to greater RRs.[2, 8, 10] If increased amounts of analyte are desired, a longer
collection time can be employed. If a short collection time is desired, an experiment will detect
NPs reliably only if the RR is improved by other means,[7] or a more sensitive detection
technique is employed.
Progress has been made in using highly sensitive and specific detection methods for NPs
in samples obtained via microdialysis, relying mainly on mass spectrometry (MS) [1, 3, 4, 1113]. Some of these studies use MS for surveys of NP content and identity [14-23]. Other studies
use MS for quantitation of identified NPs in microdialysate, mostly with selected reaction
monitoring (SRM) of daughter or granddaughter ions [2, 10, 24-30]. Finally, microdialysates can
be analyzed via MS-based techniques for NP discovery combined with less precise quantification
methods commonly used in proteomics [31-36]. In addition to MS-based analysis of dialysates,
other sensitive techniques, including those that rely on immunochemical or spectrophotometric
detection, have been used for quantitation of NPs in dialysates, but these methods lack specificity
[1, 3, 13]. Although MS instruments are highly sensitive, not all perform adequately in the
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concentration range at which NPs are present in vivo, and other methods to increase sensitivity
must be investigated.
An important method to increase the sensitivity of NP detection in MD is to increase the
RR. The relative recovery can be increased by adding affinity agents (AAs) to the liquid
perfusing the probe. The analytes form interactions with the AAs, thus increasing the quasiequilibrium driving force for further analyte to diffuse into the probe [7]. This technique is
termed affinity-enhanced microdialysis (AE-MD), and has been used by a number of different
researchers with a wide variety of compounds [5-7, 17, 34, 35, 37-52]. Stenken and colleagues,
among others, have achieved success in improving the recovery of cytokines in vitro and in vivo
[7, 39, 51, 53, 54] and neuropeptides in vitro [43], using free antibody, cyclodextrins, and
micron-sized beads coated with antibodies or heparin.
AE-MD is not yet optimal, as saturation of the beads can occur, leading to non-linear
recovery enhancement. Clogging or settling of the beads in solution is also a major concern. The
technique has also not yet been applied to study NPs in vivo (although the cytokine CCL2 has
been studied in rats using AE-MD [54]), nor have smaller beads been employed as affinity
agents. In this work, several AAs are tested for enhancement of NP recovery. Nanoscale
magnetic beads are developed for use as AAs, with the advantages of reduced settling rate and
greater binding capacity. They enhance recovery of 4 out of 6 NP standards tested in vitro. They
are also employed in vivo to study the time course of NP release following feeding in the Jonah
crab, Cancer borealis. This new affinity agent for AE-MD greatly increases the utility of this
technique for monitoring peptide secretion during behavior.
4.3. Materials and Methods
More detail for this section can be found in Appendix A.
4.3.1. Reagents
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Peptide standards (bradykinin (BK), somatostatin-14 (SMT), substance P (SP), Homarus
americanus FMRFamide-like peptide I (FLP I), H. americanus FMRFamide-like peptide II (FLP
II), and FMRFamide) were purchased from American Peptide Co. (Sunnyvale, CA, USA) and
used without further purification. C18 silica microparticles (C18SµP) were purchased from
Varian (now Agilent Technologies, Santa Clara, CA, USA) and were 5µm in size with 300Å
pore size. They were used in perfusate at a concentration of 0.2mg/mL, or 3.04 x 103 beads/µL.
C18 magnetic microparticles of 1µm diameter (C18MµP) were purchased from Varian at a stock
concentration of 2 x 106 beads/µL and used in perfusate at 3.3 x 104 beads/µL Magnetic
microparticles pre-coated with protein G were purchased from New England Biolabs (Ipswich,
MA, USA), with a stock concentration of 3.11 x 104 beads/µL and a final perfusate concentration
of 518 beads/µL. Magnetic nanoparticles of 100nm diameter pre-coated with protein G were
purchased from Chemicell GmbH (Berlin, Germany) with a stock concentration of 1.8 x 1010
beads/µL, and thus perfusate concentrations of 3.0 x 108 beads/µL and 1.8 x 109 beads/µL as
indicated below. Bovine serum albumin (BSA) and formic acid (FA) were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Polyclonal rabbit anti-FMRFa antibody was purchased
from Abcam (Cambridge, MA, USA). All other chemicals were purchased from Fisher Scientific
(Pittsburgh, PA, USA) at ACS reagent-grade and used without further purification. ACS reagentgrade solvents and Milli-Q water were used for sample preparation. Optima grade solvents were
used for operation of the UPLC-QTOF. C18-coated magnetic beads and antibody-linked beads
were prepared and used as recommended by the manufacturers. Details of preparation,
unbinding, and in vitro bead binding assays can be found in the supplementary information.
4.3.2. Animals
Jonah crabs (Cancer borealis) were purchased from Ocean Resources, Inc. (Sedgwick,
ME, USA) and The Fresh Lobster Company (Gloucester, MA, USA). These crabs were wild-
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caught and shipped overnight packed on ice. The crabs were then maintained in an artificial
seawater tank at 10-12°C, with crushed gravel as a substrate. The water from two 55 gal and one
40 gal tanks was combined and continually recirculated through a filtration apparatus (total
system volume ~160 gal) with a protein skimmer and water chiller. The tank system was
modified from a system purchased previously from Aquatic Eco-Systems, Inc. (Apopka, FL,
USA). Levels of salinity, dissolved oxygen, nitrate, nitrite, pH, and ammonia were checked
regularly and adjusted with commercial aquarium maintenance products or by adjusting filtration
settings. The room was kept on a 12hr/12hr dark/light schedule, with the dark photoperiod from
10AM to 10PM so animals could be observed during their normal active period.
4.3.3. Microdialysis Supplies
CMA/20 Elite probes with 4 mm membranes of polyarylether sulfone (PAES) were
purchased from CMA Microdialysis (Harvard Apparatus, Holliston, MA, USA). All MD probes
were rinsed with water prior to use. Several pumps were used, including a CMA/102, a KD
Scientific 100 (KD Scientific Inc., Holliston, MA, USA), and a Harvard 22 (Harvard Apparatus,
Holliston, MA, USA). When required, additional FEP (CMA) or PEEK (Upchurch-Scientific,
Idex Health and Science, Oak Harbor, WA, USA) tubing was connected to the tubing of the
probe by flanged connectors from CMA, and BASi (West Lafayette, IN, USA). BD (Franklin
Lakes, NJ, USA) plastic syringes were typically used. Flanged connectors were used to connect
21 gauge Luer-lock needles (included with CMA 20 series probes) blunted by grinding with a
rotary tool (Dremel, Robert Bosch LLC, Farmington Hills, MI, USA) to the probe tubing.
4.3.4. In Vitro MD Experiments
For in vitro experiments, the tip of the probe was immersed into a vial with a home-built
apparatus to hold the probe in place. Typically, 3 different probes’ tips were immersed in the
solution in the vial concurrently to provide multiple experimental replicates. The vial contained
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microdialysis medium, which consisted of a phosphate buffered saline (PBS) solution with
neuropeptide standards of interest dissolved in it at known concentrations, in the 1-5 micromolar
range, except for C18 silica particles, which used 50µM. The vial with probe holder was placed
on an orbital rotating platform to produce constant mixing. A sample of the medium was taken
prior to starting microdialysis and following the experiment. The probe was allowed to
equilibrate at the flow rate of the experiment (0.5 µL/min) for 30 min before starting dialysate
collection. Technical replicates were taken as consecutive 30 min samples of the liquid flowing
out of the tubing. Samples from the medium taken before and after the experiment were used to
determine the relative recovery percentage. A minimum of three technical replicates were
obtained per experiment, and a minimum of three experimental replicates were obtained, each
coming from either a different probe or a different instance of setting up and conducting the
experiment. Medium samples and samples containing no AA were placed immediately in a 96well sample plate for UPLC-QTOF analysis. For AE-MD, NPs were unbound from AAs as
recommended by the manufacturer and combined with the liquid portion of the sample in a 96well plate for analysis. The percent of beads passing through the probe was determined by
counting on a hemacytometer for micron-sized beads, and by comparing the dry mass of
particles for nanoscale beads.
4.3.4.1. Preparation and use of Magnetic Beads
C18 silica microparticles (C18SµP) were dissolved in water with 0.5% BSA to aid
solubility and 0.1% FA to improve NP binding to the beads. Following MD, the beads were
separated from the solution by employing paper filter spin cups that fit into 1.5mL tubes. The
solution containing the solid particles is loaded into the cup, which is inserted into a tube. The
tube is then centrifuged, upon which the liquid flows through the filter into the tube, and the
solid particles are retained. For MD experiments, the flow-through was saved and added to the
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liquid recovered from washing the beads in order to collect all analytes present in the dialysate.
Analyte was eluted from the C18SµP by incubating them with a solution of 50/50/0.1
ACN/water/formic acid (FA) and centrifuging. The flow-through was recombined with the initial
flow-through and solvent evaporated to dryness with a SpeedVac (Savant SC 110, Thermo). It
was then resuspended in 0.1% FA and placed in UPLC vials for further analysis. A similar
procedure was used for C18 magnetic microparticles, with the exceptions of using a magnet
instead of a spin cup for separating solid particles and not adding BSA to the perfusate.
All magnetic beads in this study were separated from the solution by holding a
neodymium magnet to the side of the vial for 30 sec followed by pipetting out the liquid. Protein
G magnetic microbeads were linked to anti-FMRFa antibody following a procedure similar to
that presented by the manufacturer. Briefly, the beads were rinsed several times with a phosphate
buffered saline (PBS) solution and incubated with shaking at 4º C overnight with the antibody at
a dilution of 1:500 (ratio of 50µL beads: 1µL of a 1:10 dilution of serum). The beads were then
again rinsed and stored in PBS. These are termed antibody-coated magnetic microparticles,
abbreviated AbMµP. For in vitro assessments of bead-binding efficiency, 30µL of a solution
containing neuropeptide standards in the 10-5-10-6M range was incubated with 30µL of beads for
30 min. The beads were then washed with PBS to remove unbound compounds, and this flowthrough was saved for analysis. Unbinding of the analytes from the beads was achieved by
incubating them with 2% formic acid (FA) at room temperature for 10 min.
Protein G magnetic nanobeads were prepared in much the same way as AbMµP, with the
exception of shorter incubation times for all steps and a second concentration for antibody-bead
incubation, as recommended by the manufacturer. The same antibody concentration used with
the microbeads was employed, 1:500, and these are termed antibody-coated magnetic
nanoparticles (AbMnP). In addition, an antibody concentration ten times higher (1:50) was
183
employed in vivo and in bead-binding assays, as this was the appropriate antibody concentration
indicated by the nanobead manufacturer. Incubation with the antibody was conducted for 15 min
at room temperature, and incubation with the test solution for in vitro binding assays was carried
out for 15 min. The unbinding step involved 2 min incubation at room temperature with 2% FA
in water.
C18 magnetic microparticles (C18MµP) were washed with PBS prior to use. Elution was
conducted by incubating the beads with a solution of 50/50/0.1 ACN/water/FA for 2 min at room
temperature. For desalting physiological samples, the solution containing NPs was dissolved in
0.1% FA and incubated with the beads for 2 min at room temperature. They were then washed 3
times with 0.1% FA. The NPs were eluted from the beads twice with the 50/50/0.1
ACN/water/FA solution into UPLC vials, and additional water (to reduce the strength of the
injection solvent) and a known amount of bradykinin was added.
4.3.4.2. Affinity-Enhanced MD In Vitro
When affinity agent was used, a clean steel ball bearing of appropriate size (1/8 inch,
Wheels Manufacturing, Louisville, CO, USA) was added inside the barrel of the syringe
delivering microdialysate. The pump was placed into a rocking platform shaker with the syringe
placed at an angle to the axis of rotation of the shaker. The rolling of the ball bearing inside the
syringe kept the affinity agent in solution [55]. For the affinity agent perfusate, the equivalent of
50µL of bead solution was diluted to 3mL with PBS (a dilution of 1:60), with one exception.
Although the concentrations of beads in mg/mL varied, it was determined that they had equal
activity per mL, as the manufacturers’ protocols recommended the same ratio of beads to sample,
i.e. 50µL beads with 0.5 mL cell lysate, a 1:10 ratio. In one set of experiments, a higher
concentration of affinity agent was used, as it was possible to increase bead concentration
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without adverse experimental effects. This trial is noted as 6x AbMnP, containing six times as
many nanoparticles per unit volume (50µL of bead solution diluted to 0.5mL of perfusate).
4.3.5. In Vivo Microdialysis
The procedure for implantation of a MD probe was adapted from previous publications
[14, 15]. A Jonah crab was anesthetized for 15 min on ice. The crab’s shell in the area
immediately above the pericardial sinus was washed with 30% bleach followed by ethanol. It
was then scored in diagonal lines using a grinding wheel on a rotary tool. A small amount of hot
glue was applied to the MD probe to mark the deepest point to which the probe would be
inserted. A 1/32” hole was drilled in a location estimated to be immediately above the heart, and
the probe was inserted. Loctite super glue gel (Loctite, Westlake, OH, USA) was applied in a
small circle around the probe. Mighty Putty (epoxy materials suspended in a clay-like base,
Mighty Putty, North Wales, PA, USA) was applied on top of this glue circle and then formed
into a cone shape around the exposed part of the probe. Any remaining voids were filled with
super glue gel. The crab was kept on ice until the glue was no longer pliable (10-15 min) and
then replaced in the tank. The day of surgery was designated as day 1 and further experiments
were conducted following a surgical recovery period of 2 days.
4.3.5.1. In Vivo MD feeding study
The probe was surgically implanted in the crab 2 days prior to the first feeding
experiment, and the last feeding experiment was conducted 8 days after surgery. This time
window was chosen to avoid effects from surgery (stress of anesthesia and being out of water,
trauma to the hypodermis) and tissue growth over the probe’s active membrane. Artificial crab
saline (440 mM NaCl;11 mM KCl; 13 mM CaCl2; 26 mM MgCl2; 10 mM HEPES acid, pH 7.4)
was used as the basis for perfusate. The flow rate was 0.5 µL/min, supplied by a programmable
syringe pump (KD Scientific Model 100, Holliston, MA, USA) and samples were collected
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every 30 min with a refrigerated fraction collector (BASi Honeycomb, Bioanalytical Systems,
Inc. Indianapolis, IN, USA). The dead volume of the system from the probe tip to the collection
tube was estimated to be 30µL, equal to 60 min at our flow rate. Flow was initiated through the
system 30 minutes before feeding the animal. The first 30 min fraction was discarded as it was
collected while the probe was not yet in equilibrium with its surroundings. Collection of feedingrelated samples was begun and the animal was fed. Samples were collected every 30 min up to
180 min post-feeding. The first two samples, taken before the animal was presented with food,
were designated as baseline samples. Typical MD was conducted on days 3, 5, and 6, and AEMD using AbMnP was conducted on day 8. For AE-MD in vivo, a 1:10 dilution of nanobeads
was used (equal to the high AbMnP concentration for in vitro AE-MD studies). Upon collection,
1.5 µL of formic acid was added to each sample to improve NP stability [2] and unbind NPs
from the antibody-coated nanoparticles, and an internal standard (bradykinin, 1µM) was added
for quantitation. Samples collected without affinity agent were directly injected onto the UPLCMS system, and magnetic beads were removed from AE-MD samples prior to addition of
internal standard and MS analysis.
4.3.6. UPLC-MS and UPLC-MS/MS Analysis and Data Processing
In vitro MD samples were analyzed via a UPLC-MS approach. A Waters nanoAcquity
UPLC system (Waters, Millford, MA, USA) was used in conjunction with a home-packed
capillary column (360 µm OD, 75 µm ID, 10cm long, Magic C18 particles (Michrom, Auburn,
CA, USA), 3 µm diameter, 100Å pore size) with integrated laser-pulled (approx. 7 µm diameter,
with a Sutter Instruments P-2000 (Novato, CA, USA)) ESI emitter tip. This was coupled directly
to a Micromass (currently Waters) QTOF micro with capillary voltage of 3500 V run in MS only
mode over the mass range m/z 500-700. Medium samples flanked dialysate samples. Table 5.1
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enumerates the retention times of the peptides using a reverse phase separation (H2O/ACN/0.1%
formic acid) with gradient from 95% aqueous to 95% organic over 30 min.
For quantitation, QuanLynx software (Waters) was employed. The extracted ion
chromatogram (XIC) for the dominant peak of each of the 6 NPs was generated and the area
under the curve was calculated. These were all divided by the total ion current (TIC) value for
that chromatogram to normalize for ion response variations throughout the run. Percent recovery
was calculated as the corrected area divided by the average value obtained from the two medium
samples. Statistical significance was determined using JMP statistical software (Version 9.0.2
SAS Institute, Inc., Cary, NC, USA). For data obtained using C18 silica microparticles, a
student’s t-test was conducted. For the remaining data, one-way analysis of variance (ANOVA)
tests were conducted, followed by post-hoc Tukey-Kramer honestly significant difference (HSD)
tests with α=0.05. Means, standard errors, and p-values are reported. Graphs illustrating this data
were generated in Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA).
The linear quantitation range of the UPLC-MS system in use was also determined by
creating a series of samples containing FLP I and FLP II at a range of concentrations with a
constant amount of BK. These samples were analyzed via the same method and linear regrssions
were generated in Microsoft Excel.
In vivo MD samples were analyzed using the same instrumentation platform but with
different MS and LC specifications. The LC gradient was 60 min long and a larger MS window
was monitored for quantitative analyses. For in vivo MD samples, quantitation was also done
with QuanLynx software. QuanLynx method files were created that would quantify multiple
charge states of the NPs in C. borealis that have been previously found to change upon feeding
or previously detected in C. borealis microdialysate. A similar method was followed by these
QuanLynx files: generation of the XICs followed by integration. The values were not normalized
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to TIC, however. The mass spectra were also checked to confirm mass error of less than 150
ppm from the predicted sequence. Those peaks that were present in very low abundance (area
less than 5 counts) were not quantified. A peak had to be present in all time points with sufficient
abundance for quantitation. Areas for detected peaks were divided by the area for bradykinin in
that run. These values were then divided by the value at t=-15min to yield fold-changes.
4.4. Results and Discussion
4.4.1. In vitro recovery enhancement
Due to previous work using column packing materials as AAs [34, 35, 38, 39, 43, 45,
49], initial experiments employed C18 silica microparticles as a generic, easily obtained AA
(Figure 4.1). These experiments led to a modest increase in NP recovery, but problems in their
implementation, including bead settling/clogging (only ~25% pass through the probe and tubing)
and the need to use additives (BSA) to improve bead dispersion made them impractical for in
vivo use. In order to obtain non-specific affinity enhancement of NPs, another type of particles
that had C18 surface functionality but also magnetic cores for simplified sample handling and
other surface modifications for increased water solubility were employed, C18 magnetic
microparticles (C18MµP). These 1 µm diameter particles are commonly employed for removal
of salts from biological samples prior to analyses that are sensitive to salt, such as mass
spectrometry. Results obtained using C18MµP as affinity agents are presented in Fig. 4.2 and
Table 4.2. The C18MµP significantly enhanced the recovery of 4 peptides—FLP I, FLP II, SP,
and SMT. Recovery was at least doubled, with final RRs of several compounds at 50% or higher.
However, settling and clogging were still observed due in part to the propensity of the particles
to attract each other via magnetism. The settling observed was less than the C18 silica particles
due to surface modifications of these particles for improved aqueous solubility.
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Based on previous work employing antibody-coated microspheres [6, 7, 37, 39, 41-43] to
improve the recovery of cytokines and neuropeptides, antibody-coated microparticles were also
employed for AE-MD. Commercial magnetic immunoprecipitation (IP) kits and a commercially
available anti-FMRFa antibody were used to create antibody-coated beads. Traditional agarose
bead-based IP kits contain beads of ~140 µm diameter, which is unsuitable for passage through
the probe and tubing. Magnetic IP kits employ smaller beads (diameters 1-5 µm), and have the
advantage of simpler separation. Magnetic microparticles coated with protein G were linked to
rabbit polyclonal anti-FMRFa as recommended by the manufacturer to create antibody-coated
magnetic microparticles (AbMµP) and added to the perfusate.
The relative recoveries obtained with and without AbMµP in the perfusate are
enumerated in Fig. 4.3 and Table 4.2. No significant increases in RR were obtained for the NP
standards, with the exception of SMT, whose RR was enhanced significantly (p=0.002) by about
2.5-fold. These results do not mirror findings of in vitro bead-binding assays (Figure 4.4), in
which SMT bound poorly to AbMµP and other NPs had a high degree of binding. Bead settling
and tube clogging were observed to a similar extent as observed with the magnetic C18 particles,
and this could explain these contrary results—beads with bound NPs may have remained stuck in
the tubing. Approximately half of the AbMµP passed through the probe and tubing.
Magnetic nanoparticles of 100 nm diameter were the final affinity agent tested in this
study. These particles are also coated with protein G and were conjugated to the same antiFMRFamide antibody (AbMnP). Particles of this size do not have permanent magnetic fields and
thus are not attracted to each other in the absence of an external magnetic field [56]. This greatly
reduces settling of the beads in the syringe and tubing. The smaller size of these particles also
reduces settling, and it was observed that around 100% of the particles pass through the tubing.
Two different concentrations of nanoparticles were used; one that was equal to that used for the
189
magnetic microparticles and C18 beads, and one that was 6 times concentrated (6x AbMnP),
possible due to the reduced settling and lack of magnetic interaction between nanoparticles.
RR enhancements are shown in Fig. 4.5 and Table 4.2. Statistically significant (p<0.05)
recovery enhancements were obtained with AbMnP for 3 of the 6 NPs. When the concentration
of nanobeads was increased, 4 out of 6 NPs had significantly enhanced RRs. FMRFa showed a
strong trend toward enhanced recovery, but this trend did not meet the statistical significance
threshold. The recovery of bradykinin was not enhanced by any of the affinity agents, due likely
to its small, hydrophilic character and lack of an amidated C-terminus. From the data obtained
here for several NPs with different sequences, it is fair to assume that the antibody used
primarily recognizes C-terminal amidation, followed by hydrophobic and basic amino acids. As
a side note, the promiscuity of this antibody provides additional support for the use of mass
spectrometry as an unbiased technique for NP analysis.
One concern that thus becomes important due to non-specific binding to antibodies is
saturation of the beads’ binding capacity. High-concentration components of a complex
biological sample (or fragments thereof), such as albumin in mammals and cyanin proteins in
crustaceans, could fully occupy these sites in vivo, and thus binding of low-concentration
biological molecules of interest to the beads will be non-linear and unreliable for accurate
representation of in vivo concentration changes. Thus, antibody-linked beads in microdialysis
perfusate should be used with caution when attempting to accurately determine the concentration
changes of analytes in the extracellular environment. In addition, if a compound is to be
delivered to an animal via MD, it may bind to the affinity agent and reduce the overall dose of
the compound delivered.
4.4.2. In vivo recovery enhancement
190
Several proof-of-principle tests to determine the suitability of affinity-enhanced
microdialysis for in vivo application were conducted. Jonah crabs (Cancer borealis) were
implanted with microdialysis probes following a modified version of a published technique [14].
The probes of several crabs were perfused with crab saline or antibody-linked nanobeads in crab
saline solution.
Representative data for baseline NP content in microdialysis samples obtained with and
without AbMnP in the perfusate is shown in Fig. 4.6. Here, extracted ion chromatograms (XICs)
for two FMRFamide-like peptides in samples obtained under baseline conditions with and
without AbMnP are displayed. These samples were obtained from the same crab on different
days, with the AbMnP experiment conducted on the last day, so probe fouling is not a factor.
They were also analyzed on the same day after storage under high acid conditions, which have
been shown to stabilize MD samples [2]. XICs are plotted with the same y-axis scale after
smoothing and baseline subtraction. FLP peaks from samples obtained without AbMnP have
intensities that are 20 to 40% as intense as those obtained with AbMnP. Only very slight
retention time shifts are observed, likely due to changes in ambient temperature. The UPLC
column was kept at room temperature, which varies several degrees throughout the day. A
similar qualitative enhancement of peak intensity was observed in two other AE-MD
experiments, conducted on different crabs, the results of which are not presented here.
Quantitative analysis also indicates that AbMnP improve NP recovery in vivo. Table 4.3
enumerates NPs previously detected in MD and whether these compounds were also detected in
samples obtained during a feeding experiment with or without AbMnP in the perfusate.
Compared with previous work in which only 35 NPs were detected in samples collected over 418 hours and concentrated ~100-fold prior to analysis [14], the detection of 31 NPs in a sample
collected over only 30 min and analyzed without preconcentration is a great enhancement in
191
sensitivity. Microdialysate from the same crab obtained with simple crab saline as perfusate only
contained an average of 25 NPs, with 17 of those detected in all three feeding trials. Therefore,
the increased NP detection sensitivity is due not only to enhancements in UPLC-MS sensitivity
that have occurred since the previous work was published, but also to the affinity-enhanced
recovery by AbMnPs.
For many trials without affinity agent in the perfusate, it was possible to detect NPs with
reasonable reproducibility across trials, but their abundance was so low that reliable quantity
changes could not be observed. In other words, the NPs were present at their lower limit of
detection (LLOD), which is below their lower limit of quantification (LLOQ). Addition of
affinity agents increased the concentration of the NPs for collection and detection to a higher
level, and thus reliable quantitation could be conducted. While concerns about AA active site
saturation should still be addressed by validation of observed NP trends by more sensitive
techniques, affinity enhancement improves MD from a mostly qualitative technique to a
quantitative technique by increasing the concentration of NPs to above their LLOQ. The LLOQ,
LLOD, and linear range of this detection method when used with BK as an internal standard are
illustrated in Fig. 4.7. For the LC-MS system employed, the LLOQ of these peptides is around
45 µM, and the LLOD is ~5 µM. Thus, NPs must be enriched for reliable quantitation.
AE-MD was employed to enrich NPs collected in vivo. Results are indicated in Fig. 4.8,
which describes relative concentration changes in seven identified crab NPs following feeding.
All data points were obtained from the same crab; three feeding trials were conducted without
AA, and one was conducted with AbMnP. The means and SEMs are plotted for the no AA trials.
The NPs shown had low variance between the three no AA trials, and were detected in all time
points. However, their concentrations do not appreciably change as they cannot be reliably
quantified. Dynamic changes in these peptides are observed when AbMnPs are added to the
192
perfusate, increasing the concentration of the NPs to a level at which they can be quantitated.
This result has been replicated in a separate C. borealis feeding trial (unpublished data).
Most of the NPs appear to increase in concentration following feeding; reaching peak
levels around 105 min after feeding. It is possible, therefore, that these NPs are neurochemicals
involved in feeding. In the authors’ experience, crabs start feeding immediately after food is
presented and stop 15-30 min. later, while still holding on to the food. They then recommence
eating a few minutes later, around 60-90 min. after food is presented. This may occur as a result
of the crab filling its stomach with food, allowing that food to be processed by the stomach and
passed on to more distal parts of the digestive tract, and followed by repetition of the process.
Since some of the NPs gradually increase before reaching peak concentration (RYLPT,
SDRNFLRFa, YSFGLa, and pERPYSFGLa), these peptides might be continually released into
the hemolymph while the animal is eating, and reaching a certain peak level could be an
indicator of satiety. Other NPs that have a more dramatic spike in concentration levels
(GPRNFLRFa, I/LNFTHKFa, and NFDEIDRSGFGFN) could be released after satiety is
reached. Although these results are preliminary and further investigation will be required, they
demonstrate how AE-MD can be used to observed dynamic changes in NPs that would otherwise
appear unchanged in typical MD experiments, due to their presence below their LLOQ.
4.5. Conclusions and Future Work
AE-MD has been conducted with a variety of AAs. AbMnP provided the greatest
enhancement in neuropeptide recovery and could be used at higher concentrations than AbMµP
due to their non-aggregation. This permitted statistically significant recovery enhancements for 4
out of 6 NP standards tested. The recovery enhancement was not specific to compounds with
sequence similarity to FMRFamide, the compound against which the antibody was generated,
and thus the mechanism of recovery enhancement is non-specific. This is likely due to the high
193
cross-reactivity of most NP antibodies, and although it could be detrimental if one desires to
enrich a single NP for analysis due to specific interest in that compound, it will provide greater
success in NP discovery and survey experiments, in which multiple and/or unknown NPs are of
interest. These AbMnPs were used in experiments in the crab to determine the roles of
circulatory NPs in feeding. They increased NP identification and made quantitation of NPs
possible during a dynamic process. This will enable assignment of putative function for several
NPs in feeding. AE-MD with AbMnP is a technique that has great potential to enrich NPs in
microdialysate for correlation of function with molecular identity.
4.6. Respective Contributions and Acknowledgements
CMS designed and carried out experiments. LL designed experiments and advised. CMS
and LL wrote the paper. Andrew M. Kozicki is acknowledged for assistance with antibody beadbinding assays. Kevin T. Hayes is acknowledged for data processing assistance in the MD
feeding study.
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50. Trickler, W. J.; Miller, D. W. Use of osmotic agents in microdialysis studies to improve the recovery of
macromolecules. J Pharm Sci. 2003, 92, 1419-1427.
51. Wang, Y.; Stenken, J. A. Affinity-based microdialysis sampling using heparin for in vitro collection of human
cytokines. Anal Chim Acta. 2009, 651, 105-111.
52. Ward, K. W.; Medina, S. J.; Portelli, S. T.; Mahar Doan, K. M.; Spengler, M. D.; Ben, M. M.; Lundberg, D.;
Levy, M. A.; Chen, E. P. Enhancement of in vitro and in vivo microdialysis recovery of SB-265123 using
Intralipid and Encapsin as perfusates. Biopharm Drug Dispos. 2003, 24, 17-25.
53. Duo, J.; Stenken, J. A. Heparin-immobilized microspheres for the capture of cytokines. Anal Bioanal Chem.
2011, 399, 773-782.
54. Duo, J.; Stenken, J. A. In vitro and in vivo affinity microdialysis sampling of cytokines using heparinimmobilized microspheres. Anal Bioanal Chem. 2011, 399, 783-793.
55. Cooper, R.; Lee, L. Chips & Tips: Preventing suspension settling during injection. Lab on a Chip [serial on the
Internet]. 2007: Available from:
http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/suspension_injection.asp#.
56. Safarik, I.; Safarikova, M. Magnetic techniques for the isolation and purification of proteins and peptides.
BioMagnetic Research and Technology. 2004, 2, 7.
196
4.8. Figures
100.0%
AE-MD with C18
Magnetic
80.0%
Microparticles
No AA
60.0%
p=0.0093
Fold-change:1.29
C18SµP
40.0%
20.0%
0.0%
FLP I
Figure 4.1. Recovery enhancement of FLP I by adding C18 silica microspheres to the perfusate.
The experiment was carried out 3 times and the mean recoveries are indicated with error bars
showing the SEM. The p-value for a Student’s t-test and the fold change are indicated on the
graph. Abbreviations are as follows: Homarus americanus FMRFamide-like peptide I (FLP I),
C18 silica microparticles (C18SµP), and no affinity agent (No AA).
100.0%
80.0%
AE-MD with C18 Magnetic
Microparticles
*
* No AA
60.0%
C18MµP
*
40.0%
*
20.0%
0.0%
FMRFa FLP I FLP II
SP
SMT
BK
Figure 4.2. Relative recovery enhancement for six different NP standards by the addition of C18
magnetic microparticles to the perfusate. Values indicated are the means, with error bars
showing the SEM. NP names are abbreviated as follows: Homarus americanus FMRFamide-like
peptide I (FLP I), Homarus americanus FMRFamide-like peptide II (FLP II), substance P (SP),
197
somatostatin-14 (SMT), and bradykinin (BK). C18 magnetic microparticles are abbreviated
C18MµP, and no affinity agent is written as No AA. Significant differences (p<0.05) from the
No AA condition are indicated with an asterisk (*).
100.0%
80.0%
60.0%
AE-MD with Antibody-Coated
Microparticles
No AA
AbMµP
*
40.0%
20.0%
0.0%
FMRFa FLP I FLP II
SP
SMT
BK
Figure 4.3. Relative recovery enhancement for 6 NP standards by the addition of antibodycoated magnetic microparticles to the probe perfusate. Values indicated are the means, with error
bars showing the SEM. NP abbreviations are indicated in the legend to Fig. 1. Antibody-coated
magnetic microparticles is abbreviated AbMµP, and no affinity agent is written as No AA.
Significant differences (p<0.05) from the No AA condition are indicated with an asterisk (*).
198
120.0%
90.0%
Microbeads
A.
unbound
B.
Nanobeads 1:500
80.0%
bound
100.0%
unbound
bound
70.0%
60.0%
80.0%
50.0%
60.0%
40.0%
40.0%
30.0%
20.0%
20.0%
10.0%
0.0%
0.0%
bradykinin HoA FLP HoA FLP
II
I
SMT
FMRFa
Nanobeads 1:50
120.0%
100.0%
SP
C.
unbound
bound
80.0%
60.0%
40.0%
20.0%
0.0%
Figure 4.4 Bound and unbound percentages of NPs in bead binding assays for antibody-linked
particles. A) Microscale beads. B) and C) Nanobeads prepared by incubating with antibody at a
B) 1:500 or C) 1:50 dilution. Abbreviations in text. Each incubation was carried out 3 times and
the average values for these are indicated with SEM as the error bars.
199
100.0%
80.0%
60.0%
AE-MD with Antibody-Coated
Nanoparticles
*†
*†
No AA
*†
AbMnP
6x AbMnP
*
*
*
*
40.0%
20.0%
0.0%
FMRFa FLP I FLP II
SP
SMT
BK
Figure 4.5. Relative recovery enhancement for 6 NP standards by the addition of antibodylinked magnetic nanoparticles at a concentration equivalent to those used with previous affinity
agents and at a concentration six times higher. Values indicated are the means, with error bars
showing the SEM. NP abbreviations are indicated in the legend to Fig. 1. Antibody-coated
magnetic nanoparticles is abbreviated AbMnP, the higher concentration is noted as 6x AbMnP,
and no affinity agent is written as No AA. Significant differences (p<0.05) from the No AA
condition are indicated with an asterisk (*). Significant differences (p<0.05) from the AbMnP
condition are indicated with a dagger (†).
200
A)
B)
Figure 4.6. Ultrahigh performance liquid chromatography-time-of-flight (UPLC-TOF) extracted
ion chromatograms for two peptides of interest (A. GPRNFLRFamide, B. ENRNFLRFamide, in
their +2 states) in microdialysis samples obtained from a single Cancer borealis under baseline
conditions. The highest intensity peak (red) represents a sample collected with AE-MD, with
antibody-linked magnetic nanoparticles (AbMnPs) in the perfusate, and the two lower intensity
peaks (maroon and black) represent samples collected without affinity agent from the same crab.
Samples were all run on UPLC-TOF on the same day with the same chromatographic and mass
spectrometric conditions. Chromatograms are smoothed, baseline subtracted, and plotted with
the same Y axis scale, which is represented as raw ion counts. Retention times are indicated at
the top of the peak, and the masses selected for generation of the XIC are indicated in the top
right corners.
201
FLP I
FLP II
1.5
y = 0.0136x + 0.0423
R² = 0.9762
4
3
Area FLP II/BK
Area FLP I/BK
5
2
1
0
0
100
200
300
y = 0.0049x - 0.1398
R² = 0.9902
1
0.5
0
0
400
-0.5
0.03
0.25
0.025
Area FLP II/BK
Area FLP I/BK
0.3
0.15
y = 0.0038x + 0.1558
R² = 0.8726
0.1
0.05
0
200
300
400
Concentration of FLP II (uM)
Concentration of FLP I (uM)
0.2
100
0.02
0.015
y = 0.0008x - 0.0103
R² = 0.5434
0.01
0.005
0
0
10
20
Concentration of FLP I (uM)
30
20
30
40
50
Concentration of FLP II (uM)
Figure 4.7. Linear range for peptide quantitation in this system. Different concentrations of NP
standards were spiked with 30 nM BK and the normalized areas were plotted against
concentration. The bottom two panels show the range at which NPs are expected to be present
endogenously.
202
GPRNFLRFa
I/LNFTHKFa
12
Fold-change
6
8
6
4
4
2
2
0 0
100
200
Time after feeding (min)
0 0
100
200
Time after feeding (min)
Fold-change
Fold-change
6
4
2
2
No AA
1.5
1
0.5 0
100
200
Time after feeding (min)
0
100
AE-MD
200
Time after feeding (min)
pERPYSFGLa
2
3
Fold-change
Fold-change
Time after feeding (min)
2.5
SDRNFLRFa
No
AA
AEMD
1.5
2
1
0
200
3
4
0
100
YSFGLa
NFDEIDRSGFGFN
8
0 0
6
5
4
3
2
1
0 0
-1
Fold-change
10
Fold-change
RYLPT (Proctolin)
8
100
1
0.5
200
0
0
100
200
Time after feeding (min)
Time after feeding (min)
Figure 4.8. Affinity-enhanced microdialysis permits observation of changes in NP levels
following feeding. The x-axis crosses the y-axis at a value of one, which would indicate no
change from baseline conditions. Without affinity agent, the NPs are present at levels too low for
changes to be observed. For No AA data, 3 feeding trials from the same crab are pictured as an
average with standard error as the error bars. Some error bars are obscured by the size of the
marker at each data point. The AE-MD data was obtained from the same crab in a feeding trial.
AE-MD data shows dynamic changes in NPs after feeding for several peptides, whereas the No
AA data shows little variation.
203
4.9. Tables
Table 4.1. Sequences of several peptide standards used in bead-binding assays. C-terminal
motifs are underlined. Details of the reverse phase separation method can be found in the text of
the article, experimental section. Abbreviation: FMRFamide-like peptide (FLP).
Table 4.1. Sequences, C-Terminal Motifs, and Reverse-Phase LC Retention Times of NP
Standards
Substance
Sequence
Retention Time (min)
FMRFa
FMRFa
18.25
Substance P
RPKPQQFFGLMa
20.68
Homarus americanus FLP I
SDRNFLRFa
19.17
H. americanus FLP II
TNRNFLRFa
18.85
Bradykinin
RPPGFSPFR
18.13
Somatostatin-14
AGCKNFFWKTFTSC
21.41
Table 4.2. Results of AE-MD experiments. Results from numerous in vitro microdialysis studies
are summarized in this table. Technical replicates were averaged to yield one value per
experiment, and these values were then combined to determine the average RR and standard
error of the mean (SEM) of that value. The number of experiments is shown as n. Analysis of
variance (ANOVA) tests indicated statistically significant differences in the RRs obtained across
the different AAs, and post-hoc Tukey-Kramer honestly significant difference (HSD) tests
yielded the p-values for significance indicated in the table. With one exception, only p-values of
0.05 or smaller are reported, as this was the significance threshold. Blank cells indicate nonsignificant p-values. Fold-changes were also calculated for the statistically significant values.
Neuropeptide (NP) names are abbreviated as follows: FMRFamide (FMRFa), Homarus
americanus FMRFamide-like peptide I (FLP I), Homarus americanus FMRFamide-like peptide
II (FLP II), substance P (SP), somatostatin-14 (SMT), and bradykinin (BK). AAs are abbreviated
as follows: C18 magnetic microparticles is abbreviated C18MµP, no affinity agent is written as
No AA, antibody-coated magnetic microparticles is abbreviated AbMµP, antibody-coated
204
magnetic nanoparticles is abbreviated AbMnP, and the higher concentration is noted as 6x
AbMnP.
Table 4.2. Recovery Enhancement Caused by Addition of Affinity Agents
Comparison to
Comparison to No AA
AbMnP
Average
NP
AA
n
SE
RR
Foldp
p
Fold-Change
Change
FMRFa No AA
6 21.90% 2.52%
C18MµP
5 23.00% 2.76%
AbMµP
7 18.50% 2.34%
AbMnP
4 23.00% 3.09%
6x AbMnP 3 34.80% 3.57% 0.0548
1.59
FLP I
No AA
7 26.30% 3.46%
C18MµP
6 73.10% 3.74% <0.0001 2.78
AbMµP
7 33.30% 3.46%
AbMnP
4 48.30% 4.58% 0.0072
1.84
6x AbMnP 3 55.20% 5.29% 0.0013
2.1
FLP II
No AA
6 26.00% 3.60%
C18MµP
6 61.50% 3.60% <0.0001 2.36
AbMµP
8 34.60% 3.12%
AbMnP
4 34.10% 4.41%
6x AbMnP 3 87.30% 5.09% <.0001
3.35
<.0001 2.56
SP
No AA
5 2.21%
2.67%
C18MµP
5 24.80% 2.67% 0.0002
11.2
AbMµP
5 7.82%
2.67%
AbMnP
3 69.70% 3.45% <0.0001 31.6
6x AbMnP 3 92.10% 3.45% <0.0001 41.7
0.0024 1.32
SMT
No AA
4 11.10% 4.55%
C18MµP
5 45.10% 4.07% 0.0003
4.09
AbMµP
6 40.10% 3.71% 0.001
3.63
AbMnP
4 37.90% 4.55% 0.0051
3.43
6x AbMnP 3 82.90% 5.25% <0.0001 7.5
<.0001 2.19
BK
No AA
6 51.60% 3.33%
C18MµP
5 56.20% 3.64%
AbMµP
7 47.70% 3.08%
6.16%
AbMnP
5 40.11%
%
6x AbMnP 3 45.20% 4.70%
205
Table 4.3. Comparison of NP complements detected with conventional and affinity-enhanced
microdialysis. The number of NPs detected in a reference study [14] was compared to the results
from this study. An x indicates a detected peptide. For the non-enhanced in vivo MD data
obtained in this study, peptides that were present in all 3 feeding trials were determined to be
detected.
Table 4.3. Comparison of NP Complement Detected with Conventional and
Enhanced In Vivo Microdialysis
Sampling Method
In
vivo In vivo AEMD [14]
MD
4-16
hrs
30 min
Sampling Time
Peptide Family
Peptide sequence
x
A-type allatostatin
ARPYSFGLa
x
x
DPYAFGLa
x
ERPYSFGLa
x
PDMYAFGLa
x
pERPYSFGLa
x
x
SPYAFGLa
TNFAFSPRLa
x
x
YAFGLa
x
x
YSFGLa
x
x
B-type allatostatin
NNNWSKFQGSWa
x
x
NWNKFQGSWa
x
STNWSSLRSAWa
x
TSWFKFQGSWa
Allatostatin
x
x
prohormone related DPYAFGLGKRPADL
peptide
DPYAFGLGKRPADLYEFGLa x
x
CabTRPs
APSGFLGM(O)Ra
x
APSGFLGMRa
x
x
APSGFQa
x
x
GFLGMRa
x
Orcokinin
NFDEIDRSGFa
x
NFDEIDRSGFG
x
x
NFDEIDRSGFGF
x
NFDEIDRSGFGFH
Affinity-
In vivo
MD
30 min
x
x
x
x
x
x
x
x
x
x
206
FLP
RYamide
YRamide
SIFamide
Other NP
NFDEIDRSGFGFN
NFDEIDRSGFGFV
NFDEIDRSSFGFN
NFDEIDRSSFGFV
APQRNFLRFa
APRNFLRFa
DGGRNFLRFa
DRNFLRFa
GAHKNYLRFa
GGRNFLRFa
GPRNFLRFa
RNFLRFa
RSFLRFa
SDRNFLRFa
SENRNFLRFa
SGRNFLRFa
SMPSLRLRFa
TNRNFLRFa
FVGGSRYa
HIGSLYRa
GYRKPPFNGSIFa
I/LNFTHKFa
PFCNAFTGCa (CCAP)
pQLNFSPGWa (RPCH)
TNFAFSPRLa (CabPK-I)
Total
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
35
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
31
17
207
Chapter 5: MS/MS Characterization of the Neuropeptidome of the Crayfish Orconectes rusticus
Adapted from Schmerberg, C. M., Liang, Z., and Li, L. J. Proteome Res. 2012 [in preparation]
5.1. Abstract
The rusty crayfish, Orconectes rusticus, is an important species in behavioral neuroscience and
invasive species ecology. It presents several well-defined behaviors, permitting it to be both an ideal
model species for studying social dominance and aggression, and a successful invasive species in the
upper Midwest United States. Monoamine neurotransmitters (NTs), human drugs of abuse, and spatial
learning have also been studied in O. rusticus. Despite its significance, the neuropeptides (NPs) of O.
rusticus have not yet been cataloged, although NPs have been implicated in aggression, addiction, and
adaptation to environmental change in other species. NP studies in other crayfish species have been
limited in the techniques or organs used. In this work, the premier technique for NP identification—
tandem mass spectrometry (MS/MS)—was used to determine the NP complements of the sinus gland
(SG) and brain in Orconectes rusticus. Fractions from high performance liquid chromatography
(HPLC) were enzymatically digested if necessary and injected onto ultra-high performance liquid
chromatography (UPLC) coupled on-line with an electrospray ionization (ESI) quadrupole time-offlight (QTOF) mass spectrometer operating in data-dependent acquisition (DDA) mode. MS/MS
interpretation was conducted by two different strategies: database search software (Mascot), and de
novo sequencing software (PEAKS). These approaches were aided by the construction of a home-built
crustacean neuropeptide database. Highly confident identifications were made for 98 previously
identified neuropeptide sequences from 8 families. Over 47 additional putative neuropeptides were
also identified from 12 families, including sequences from families already observed in Orconectes
species (CPRPs, PDHs), and peptides not previously identified in crustaceans using mass
spectrometry. These represent potentially novel neuropeptides in crustacean species. Due to overlap in
208
NP sequences, the minimum number of unique peptides that could generate the observed PSMs is 23.
This catalogue of NPs will be vital for further research into the roles of NPs in behavior,
environmental adaptation, and addiction in O. rusticus.
5.2. Introduction
The rusty crayfish Orconectes rusticus is an important species in two major areas of biology. It
is an important model species in behavioral neuroscience, studied in applications ranging from
aggression to the reinforcing effects of human drugs of abuse. Studies of O. rusticus are also of vital
importance in the field of invasive species ecology, as its invasion of several bodies of water in
Northern Wisconsin has been documented in detail.
5.2.1 Significance of Orconectes rusticus to the Field of Neurobiology
The contribution of the rusty crayfish to the field of behavioral neuroscience rests primarily on
its expression of several well-described and stereotyped behaviors, as reviewed by several authors [1,
2]. O. rusticus and other crayfish typically lead solitary lives, without alliances or pair-bonds, and
interaction is mostly agonistic, resulting from competition over resources (food, shelter, mates), but
existing even when there is no contested resource, due probably to the high density at which they live
in the wild. Social dominance is based mostly on size, with fighting determining hierarchies when the
animals’ sizes are similar. Fights have a set progression from one stereotyped behavior to another;
starting with ritualized visual displays, followed by progression to antennal whipping, claw locking,
wrestling, and finally claw use [1, 2]. The ethology of aggression in O. rusticus is thus very well
defined. Attempts have been made to understand the neurochemical basis of this behavior in the
context of monoamine neurotransmitters (NTs), as reviewed in several papers [1, 2]. Studies in
decapods including Orconectes rusticus are equivocal in the roles of amine NTs in agonistic posture
and behavior. Thus, it appears that no single NT is a whole-body signal for aggression, but that state-
209
and/or dose-specific effects are important [1, 2]. The potential of O. rusticus to serve as a model
species in the fields of addiction [3-7] and learning/memory [8-13] has also recently been
demonstrated, although the neurochemistry of these behaviors has not been studied in as much detail.
Although O. rusticus is a useful model animal in a number of behavioral neuroscience applications
and a large amount of progress has been made in understanding the neurochemical basis of this
aggression in this species, much remains to be studied.
5.2.2. Significance of Orconectes rusticus to the Field of Ecology
Orconectes rusticus is also an important species in the field of invasive species ecology. Its
invasion of the area in northern Wisconsin near the Michigan border—far from its native range in the
Ohio River drainage—has been studied in detail, with numerous research and review articles written.
A 2006 meta-review of data on crayfish collected since 1932 illustrates the displacement of several
native crayfish species by O. rusticus, which was introduced starting in the 1960’s [14]. A recent
review enumerated the factors that enable it to be a successful invader, and the negative ecological
impact it can have [15]. The aggressiveness of O. rusticus is a major factor in its ability to kill or evict
native crayfish. They decrease the diversity and amount of macrophytes, large aquatic plants that
provide both food and shelter for fish and other invertebrates. Snails and other mollusks, fish eggs, and
various types of litter are food sources for rusty crayfish, and these are reduced in number when O.
rusticus are present [15]. The potential for O. rusticus to further invade lakes and rivers and displace
native crayfish was modeled in 2011, and a 25% chance of this was determined for 115 lakes and
5,000 km of streams in Wisconsin [16]. From this and many other studies, it is clear that the rusty
crayfish presents a significant threat to freshwater ecosystems. Its survival appears to be dependent on
a threshold dissolved calcium content and pH of lake water of 2.5 mg/L and 5.5, respectively, as
reviewed by Olden et al. [14]. Predation levels also influence the population of O. rusticus [17-19].
210
5.2.3. Current Knowledge of Neuropeptide Content in Orconectes spp. and Significance of This
Work
It has previously been demonstrated that neuropeptides (NPs), an important class of
neurotransmitters with additional neurohormonal function, are important in adaptation to
environmental factors, aggression, and addiction. Although the neuropeptidomes of several other
decapod crustacean species have been determined, including the NP content in the eyestalk of the
crayfish Orconectes limosus [20], NPs in O. rusticus have not yet been described. In this work, the NP
complements of both the brain and eyestalk of O. rusticus are characterized using multiple mass
spectrometry (MS) approaches. Paramount among these is tandem MS (MS/MS), which is used to
identify the sequence of a peptide directly from the ions it produces upon gas-phase fragmentation.
This technique has become the gold standard for neuropeptide identification, as opposed to antibodybased techniques, genomics-based techniques, and sequential cleavage of amino acids (Edman
degradation). Antibody-based techniques suffer from low specificity and do not provide sequence
information. Gennomics-based techniques often cannot predict the full complexity of posttranslational modifications that NPs undergo. Edman degradation requires a large amount of highly
purified sample.
Neuropeptides are best characterized by tandem mass spectrometry (MS/MS), especially when
no genome is available, due to the large degree of post-translational processing. This work comprises
the first neuropeptide study in Orconectes rusticus. The NP contents of several ganglia in the related
species Orconectes limosus have been reported in a number of different studies, identified by MSbased techniques and/or Edman degradation, but these reports have only yielded a total of 14 unique
peptides from the following families: pigment dispersing hormones (PDH), molt-inhibiting hormones
(MIH), crustacean hyperglycemic hormones (CHH), CHH precursor-related peptides (CPRPs),
211
orcokinins, orcomyotropin, and A-type allatostatins (A-AST) [20-25]. The most comprehensive one of
the previously mentioned studies employed nanoscale liquid chromatography (nanoLC) coupled to
tandem mass spectrometry (MS/MS) for de novo sequencing of NPs obtained from an extract of the Xorgan-sinus gland (XO-SG) complex [20], one of the main neurosecretory structures in crustacean
species. This study identified a total of six peptides, with three additional truncated versions of the
CPRP peptides also identified. Compared with more recent studies in which 122 NPs could be
identified from tissues of the green crab Carcinus maenas [26], it is fair to assume that many
Orconectes NPs remain undiscovered. Advances in MS and MS/MS instrumentation, combined with
multistage separation techniques, permit such a study to provide a more comprehensive picture of the
neuropeptidome of a given species. In addition, there have been improvements in software for de novo
sequencing and MS/MS identification based on database search, and it has recently become possible to
create a database of known crustacean NPs.
In this work, the aforementioned strategies are employed to characterize the neuropeptidome of
the Orconectes rusticus XO-SG and brain (supraoesophageal ganglion). A two-dimensional reversed
phase (RP) liquid chromatography (LC) strategy was employed, with neutral and acidic mobile phases
to provide orthognality. The second dimension nanoLC was coupled directly to a highly sensitive
quadrupole time-of-flight (QTOF) mass spectrometer for MS/MS analysis. Software programs for
database search interpretation of MS/MS spectra (used in this study: Mascot, Matrix Science) and de
novo sequencing-based MS/MS interpretation (used in this study: PEAKS, Bioinformatics Solutions,
Inc.) were employed and the results are compared. In all, 145 unique peptide-spectrum matches
(PSMs) were obtained, mapping to 98 previously identified neuropeptide PSMs from 8 families and 47
putative neuropeptide PSMs from 12 families. This is the first investigation of NPs in O. rusticus and
greatly increases the number of known NPs in Orconectes and other crustacean species.
212
5.3. Experimental Section
All portions of this section can be found in Appendix A with added detail.
5.3.1. Animals
Male and female Orconectes rusticus were kindly provided by Alex Laztka, a graduate student
in the laboratory of Jake Vander Zanden, in the Department of Limnology at the University of
Wisconsin-Madison, and co-workers. They were obtained from fresh water lakes in Northern
Wisconsin centered around the Limnology Department’s Trout Lake Research Station, on Trout Lake
in Boulder Junction, WI. Animals were sampled as part of a National Science Foundation (NSF)funded Long-Term Ecological Research (LTER) study, and were obtained and transported with the
permission of the Wisconsin Department of Natural Resources. Permission for transport must be
obtained due to the status of O. rusticus as an invasive species in Wisconsin.
O. rusticus were obtained in modified minnow traps placed in lakes at a depth of
approximately 1 m, in an area where the lake substrate is “cobble,” or rocks approximately 6-8 cm in
diameter. The traps were modified by increasing the entrance hole to 5cm to accommodate mature
male crayfish. Traps were baited with beef or chicken liver. This method has been shown to
preferentially collect male crayfish, and although both genders were observed, around 75% of the
animals were male. Traps were emptied at an interval of 20-24 hrs. O. rusticus was then separated
from other catch by hand and kept in buckets in a refrigerator at 4ºC for approximately 12 hrs. The
crayfish should not be stored in water, as the water will quickly become foul and deoxygenated.
Storing animals in water in the refrigerator and/or during transport led to approximately 95% mortality
in a batch of around 350 crayfish. Stored dry and cold, they enter a state similar to hibernation and can
survive longer. They were then transferred to a carrying container that contains an ice pack and
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transported to Madison, WI by personal vehicle (a trip of approximately 3.5 hrs). Species identity was
verified prior to housing in Madison in freshwater aquaria.
Aquaria contained fresh, dechlorinated water, and crushed gravel substrate. They also were
equipped with Aquaclear 30 (Hagen) filtration units and conventional air bubblers. No more than 45
individuals were housed per 20 gallon tank. Care was taken to ensure a tight fit for the lid of the
aquarium as the crayfish can escape through very small holes. Animals were not stored more than a
day at a density of 45 animals/20 gal tank, and at lower densities, of about 20 per 20 gal tank, could be
housed indefinitely.
If kept for longer periods of time, O. rusticus were fed and monitored for molting, and regular
water changes were conducted. Crayfish will cannibalize if they are not fed every 2-3 days, and
fighting often occurs over food. They were fed white fish, as it does not foul the water and pelleted
food designed for bottom-feeding fish from a pet store. Enough food was presented so that all animals
had something to eat to prevent fighting. If O. rusticus molted, they were isolated with the old shell
until their new shell hardens. Other individuals would attack and kill unprotected newly molted
individuals. These newly molted individuals can and should consume the old shell for nutritional
purposes. Hardening takes 1-2 days. Due to waste accumulation in the substrate and water, the tanks
were cleaned weekly with a 20% water change, or more often if housed in high density.
5.3.1.1. Orconectes rusticus Dissection
Brains and eyestalks were dissected from O. rusticus without the aid of a microscope. Whole
eyestalk extracts were prepared instead of dissecting the sinus gland out of the eyestalk due to its
miniscule size, which is difficult to locate even under a dissecting microscope. O. rusticus was put on
ice until movement ceased (approximately 15-20 min). Appendages were removed and the tail nerve
cut with dissecting scissors to prevent tail flipping. The carapace was cut along a line starting below
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one eyestalk, continuing up toward the rostrum, and then down toward the other eyestalk (indicated in
figures in Appendix A). The distal part of the body was then discarded and the brain was located
inside the shell immediately behind the rostrum. It was removed with fine forceps and placed in a 1.5
mL tube containing 0.5 mL acidified methanol (90:9:1 methanol: water: acetic acid by volume) on ice.
25-30 brains were placed in a single tube. They were stored at -20°C or -80°C (if storage occurred for
more than a month) until extraction. Entire eyestalks were removed by cutting the soft tissue where
they were connected to the head with fine scissors. These were placed in a similar tube, with about 2025 pairs per tube.
5.3.2. Extraction and Sample Preparation
Tissues were homogenized in acidified methanol using plastic pestles for soft tissues and a
glass vial and pestle for whole eyestalks. Vigorous homogenization followed by sonication (5 min)
was required for all tissues, in particular the eyestalks. Centrifugation was used to separate liquid and
solid portions (5 min x 16,100 g), and the solids were re-extracted 3 times with the initial volume of
acidified methanol, saving the liquid fraction each time. It was later determined via MALDI-TOF/TOF
that the third extraction from O. rusticus brain tissue yielded mostly lipids and few peptides, so only
the first two were combined for that tissue. For other tissues, all liquid portions were combined. The
solvent was evaporated. Two brain and two eyestalk samples were prepared and analyzed, each
containing tissues pooled from 25-30 individual crayfish.
O. rusticus brain samples were subjected to a delipidation step. They were reconstituted in
water and combined with an equal volume of a solution of chloroform: methanol (3:1) and a liquidliquid extraction was conducted. The aqueous layer was saved, and the lipid layer was re-extracted 3
times. All samples were reconstituted in 0.1% formic acid (FA) in water.
5.3.3. Liquid Chromatography-Mass Spectrometry
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Samples were subjected to a first dimension of liquid chromatography as a pre-purification
step. A neutral pH (7.4, solvent A: 25mM NH4HCO2, solvent B: 9:1 ACN: 25mM NH4HCO2)
reversed-phase (Inertsil ODS-4 column, 3µm, 2.1 x 150 mm, GL Sciences, Tokyo, Japan) was used
with an Alliance HPLC (Waters, Milford, MA) system. Fractions were collected every 3 min with a
Rainin Dynamax Model FC-4 fraction collector. Early eluting fractions were combined with late
eluting fractions to reduce the total number of samples while maintaining separation.
Samples were injected onto a C18 reversed-phase column (BEH130 C18, 1.7 µm, 75 µm x 100
mm, Waters, Milford, MA) using a Waters nanoAcquity UPLC system (Waters, Milford, MA). The
outlet of this column was connected to a fused silica capillary with a pulled tip of internal diameter 5
µm (Sutter Instrument Company, Novato, CA). This was used as the ESI inlet on a Waters Synapt G2
QTOF (Waters, Milford, MA). A 120 min reversed-phase run with solvent A as 0.1% FA in water and
B as 0.1% FA in ACN was used. The instrument was operated in data-dependent MS/MS mode and
Glu-fibrinopeptide was infused for lockspray calibration.
5.3.4. Databases
A variety of approaches were used for database construction in this study. As there is no
available genome for any decapod crustacean, various databases were assembled using a combination
of in-house compiled information and publicly available data. The in-house component contained all
known crab and lobster neuropeptides, compiled from published papers out of the Li lab and others,
and theses from the Li lab. This database portion is referred to as LiDB and can be found in Appendix
B and will be made publicly available shortly. The National Center for Biotechnology Information
(NCBI) protein database (www.ncbi.nlm.nih.gov/protein) was also searched to obtain sets of relevant
known protein sequences. One such list was created by bioinformatics mining by a previous lab
member. This contained all NCBI crustacean NP sequences, several crustacean protein sequences, and
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numerous other NP sequences from a variety of multicellular organisms. This list of sequences is
referred to as WF, for the name of the previous lab member. The entire list of decapod sequences was
also downloaded from NCBI (“decapod”), as was the entire list of crustacean sequences
(“crustacean”). Finally, a database was acquired from NCBI by restricting the taxonomy to crustacea
and searching for the keywords “peptide” or “hormone” (NCBI query: (peptide[All Fields] OR
hormone[All Fields]) AND "Crustacea"[Organism]). This is referred to as crustaceanNP. Databases
were compiled in FASTA format and edited for redundancy. Only results from crustaceanNP+LiDB
are shown, as this was determined to be the most comprehensive database that did not also contain
large numbers of erroneous sequences of compounds that were either not NPs or were not from related
species.
5.3.5. Data Analysis
A home-built computer (3.30 GHz AMD FX-6100 Six-Core Processor, 32.0 GB RAM, 64-bit)
operating in Windows 7 Professional (64-bit) was used for the majority of analyses. Protein Lynx
Global Server (PLGS, Waters, Milford, MA) was used to search a variety of home-built databases
against the data obtained and to convert the data from .raw folders into .pkl files for MASCOT search.
This software also conducted an internal mass calibration with the lock mass compound. Several
databases and combinations of databases were used, as detailed in the results and discussion. For each
search, the modifications C-terminal amidation, pyroglutamation, and methionine oxidation were
always added. In all searches, a no enzyme search was conducted, a minimum of 3 fragment ion
matches per peptide were required, and mass tolerance was set to default. The software’s built-in de
novo sequencing tool was also employed with default mass tolerances and the same modifications. No
results were obtained with either the database search or de novo functions.
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The .pkl files created by PLGS were searched via an in-house Mascot server (Matrix Science,
London, UK) using the Daemon batch uploader tool. For the Mascot server, only the LiDB+WF and
LiDB+crustaceanNP databases were uploaded. For each search, the modifications C-terminal
amidation, pyroglutamation, and methionine oxidation were selected. In all searches, a no enzyme
search was conducted and mass tolerance was set to 0.5 Da for precursors and 0.25 Da for fragments.
Peptide charges were set to 2+, 3+, and 4+. Files from all fractions were combined by Mascot into a
single file for searching.
These same .pkl files were analyzed using PEAKS studio 5.3 (Bioinformatics Solutions Inc.,
Waterloo, ON). Data was preprocessed to select only high-quality spectra. De novo was conducted
with no enzyme and the modifications C-terminal amidation, pyroglutamation, and methionine
oxidation. Mass tolerance was 0.5 Da for precursors and 0.25 Da for fragments. Following the de novo
search, a comparison to database sequences was conducted. This was done with the LiDB+WF and
LiDB+crustaceanNP databases, and all other parameters the same.
5.3.6. Results Curation
Peptide spectrum matches (PSMs) obtained with PEAKS and Mascot were output in comma
separated values (.csv) format for further analysis in Microsoft Excel. Although Mascot reports a
“Mascot score,” this is their version of a -10logP value, which is also what PEAKS reports. All PSMs
scoring less than 15 were removed from the results. The two brain and two eyestalk samples were
analyzed in parallel, and the best score obtained was saved. False-discovery rates (FDRs) were not
generated by PEAKS due to the small database size, and the FDRs generated by Mascot were for a
score threshold of 40, not useful for our score cutoff of 15. Occasionally, PSMs with large numbers of
repeating residues were disregarded as likely false positives. PSMs that were assigned to proteins or to
unknown products of the Daphnia genome are listed separately in Appendix C. PSMs for peptides or
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hormones were subjected to meta-analysis to compare the performance of Mascot and PEAKS. Venn
diagrams were generated using software created at the Pacific Northwest National Laboratory. For
final reporting of the neuropeptides identified, some overlapping PSMs were manually aligned and
parsimony was used to determine the minimal number of precursors that could lead to those PSMs.
Peptides are reported in Tables 5.1-5.3 and Appendix C.
Several sequences determined by PEAKS de novo interpretation of MS/MS spectra were not
found in databases, and these were subjected to further analysis. Those that appeared in more than one
extract, or had sequence motifs common to NPs, were searched against the NCBI protein database
using tblastn and restricting to Crustacea (taxon id 6657). Matches with the lowest E-value are
reported. Where multiple proteins had the same E-value, either all are listed or they are described
generally. This data is also reported in Appendix C.
5.4. Results and Discussion
In total, 145 high-quality peptide-spectrum matches (PSMs) were made to MS/MS spectra.
These mapped to 98 peptides previously observed in decapod crustaceans using mass spectrometry,
from 8 different neuropeptide families. The additional spectra mapped to 47 putative neuropeptides
from 15 families. These either represent peptides observed in non-decapod crustaceans, or peptides
whose presence had previously only been identified by genetic techniques. Additional matches were
made to non-neuropeptide entries in the database, including hemocyanin, actin, and currently
unidentified products of the Daphnia genome. Although other databases were used, only data from
crustaceanNP+LiDB is presented, as this combination contained the most NP sequences with the
fewest non-NP or non-crustacean sequences. A smaller database is desired when searching for NPs, as
searches must be done without using specific enzyme cleavage sites to capture the full complexity of
NPs and potential novel proteolytic processing products. This smaller database, although it permitted
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such searches to be carried out in a reasonable period of time (hours instead of days), led to a number
of issues, mostly with determining the quality of a match.
Reproducibility between samples of the same tissue type was moderate. Around one third of
PSMs from Mascot and one fourth from PEAKS were observed in both pooled tissue samples (Fig.
5.1). This illustrates the necessity of analyzing multiple replicates. It is likely that this is due to the
somewhat random nature of precursor selection for MS/MS, and that technical replicates might be
sufficient. It is unlikely that the samples contained highly different NP contents, since they were
pooled tissues from 25+ animals, all collected from the same geographical region around the same
season. It is nevertheless possible that slight differences in the locations and days of collection led to
NP variation between the samples. Another possible source of this variation between samples could be
the inclusion of a number of random matches, which are not likely to occur the same way in two
different samples. This concern, however, cannot be addressed in neuropeptide studies with the current
statistical methods of assigning false discovery rates (FDRs) and the probabilities (P) of matches being
correct.
False discovery rates and match probability are determined based on statistical methods that
take into account a number of factors, and should presumably return a good estimate of the accuracy
of assigning a MS/MS spectrum to a given peptide sequence. All current methods to calculate this
value take into account the number of fragment ion matches observed versus the number of possible
matches. These formulas will provide a good estimation of the quality of a match when the peptide is
large, but may not do so for smaller peptides with fewer possible matching ions. The gold standard for
acceptability of a PSM remains visual verification, and in our experience, PSMs with lower Mascot
scores still passed this test, while PSMs with similar scores from PEAKS did not. Although both
software programs report a -10logP value, the exact scoring mechanisms to calculate that probability
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(P) differ. Therefore, it may be appropriate to use different thresholds for score cutoff when
interpreting results from each program. As indicated in Fig. 5.2, not all spectra with this score
represent good matches (A), but some do (B). PEAKS results at this probability threshold could
provide poor (C) or good (D) matches. For comparison of the programs in this study, however, a score
cutoff of 15 was chosen. This value was chosen by observations as seen in Fig. 5.2, and a desire not to
dismiss visually good matches that may have a low score. The use of crustaceanNP+LiDB for the
database also led to complications because the LiDB sequences are short, representing just the active
NP. Sequences in the crustaceanNP database are long preprohormones. Thus, matches to these longer
proteins were scored better than similar matches to the shorter active NP sequences. It is clear from
these results that the methods for assigning scores to PSMs used by both programs may not be very
accurate in describing the quality of matches to neuropeptides, and additional by-hand analysis of lowscoring matches will still be necessary.
An alternative approach that may be successful is appending decoy proteins to the database,
and choosing a score threshold based on the acceptable false discovery rate (FDR) using matches to
the decoy proteins. Mascot and PEAKS are both capable of estimating a FDR in this manner, but with
small databases that contain both neuropeptide and prepropeptide sequences, this approach may not
lead to an accurate determination of FDR. Indeed, when searching what was determined to be the
“best” database, LiDB+crustaceanNP, PEAKS did not return FDR values, and Mascot only estimated
FDR at a score of 40. The score cutoff for which Mascot returns a FDR is based on the quality of the
database, and one cannot ask the program to return a FDR for any given score, such as the score
empirically observed to produce acceptable matches (15 in this case). Therefore, another approach to
determining the number of random positive identifications of PSMs should be employed, although no
appropriate statistical method currently exists.
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5.4.1. Previously Known Crustacean Neuropeptides Identified by MS/MS in O. rusticus
Several PSMs from this study mapped to neuropeptides previously observed in other decapod
crustacean species using MS/MS. These include one B-type allatostatin (AST-B), two FMRFamidelike peptides (FLPs), myosuppressin, 16 orcokinins, orcomyotropin, and one SIFamide (see Table
5.1). PDH and CPRPs will be discussed separately, as both previously observed NPs and novel gene
products were observed. PSMs may represent 1) fragments of mature peptides that are produced as a
result of degradation or fragmentation that occurs before or after sample collection, 2) unique mature
peptides created by different processing pathways, or 3) incompletely processed peptides. Therefore,
the multitude of orcokinin sequences, most of which contain homologous sequences, could all
represent unique molecules present in the animal with unique activities. Alternatively, they could all
represent fragments of 8 individual orcokinin peptides (See Fig. 5.3.). For this reason, all PSMs are
reported. Most NPs are present in both tissues at equal abundance. This is in agreement with previous
findings in other crustacean species [20, 26-30].
Many of the identified sequences match those previously observed by MS/MS or Edman
sequencing in the closely related crayfish Orconectes limosus. Two of the orcokinins
(NFDEIDRSGFGFN and NFDEIDRSGFGFV) were previously identified in Orconectes limosus [22,
25]. A different form of orcomyotropin, FDAFTTGFamide, was previously found in the hindgut of O.
limosus [22]. Several other studies have used MS/MS or Edman sequencing techniques to map NPs in
O. limosus, including molt-inhibiting hormone (MIH) [21], A-type allatostatins [23], and CHH [24].
These peptides were not identified in this study. It would be unlikely to identify CHH or MIH using
these techniques, as they are large peptides (8-9 kDa) that require multi-scale MS approaches for
complete sequencing [31]. A typical approach would be digestion with a protease such as trypsin,
followed by MS/MS analysis of the fragments and assembling them into the full peptide, called
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bottom-up sequencing. That method was not employed in this study. The absence of A-type ASTs is
unusual and may be due to the short sequences of these peptides and the fact that many are only singly
charged. Software programs for DDA acquisition and peptide sequencing preferentially pick multiply
charged precursors, as singly charged molecules present in a LC-ESI analysis of tissue extracts are
typically lipids or small molecules as opposed to peptides. Scoring functions for both Mascot and
PEAKS are biased to produce low scores for short peptides even with good matches. These lowscoring matches may have been discarded by the software. Manual de novo sequencing of MS/MS
spectra that did not yield hits according to the software programs may be able to identify some of these
smaller peptides. It is not surprising that studies of the related crayfish O. limosus have yielded some
of the same NPs as many sequences can be expected to be similar, and the NPs that have been seen in
O. limosus but not in this study are likely missing due to different experimental or data processing
approaches.
B-type allatostatins (AST-Bs), FMRFamide-like peptides (FLPs), SIFamides, myosuppressin,
and several of the orcokinins observed have not been previously identified in the studies of Orconectes
limosus, but have been observed in many other decapod crustacean species, including other crayfish
[32-37], lobsters [38], and crabs [39-43]. Although not every species has the same set of NPs,
members of each of these families have been found in species across the order Decapoda, and some
have been found in more distantly related species as well. Of particular note is the orcokinin
DFDEIDRSGFA. This has previously been identified in one other species, Callinectes sapidus, and
may represent the product of deamidation of the typical N-terminal N residue, either in vivo or during
sample preparation and analysis [29]. One possible explanation is that deamidation of this residue is a
modification that occurs in vivo to change this orcokinin’s signaling properties. This method of
signaling modulation is not without precedent [44]. However, the binding efficiencies of
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NFDEIDRSGFA vs. DFDEIDRSGFA at their receptor have not been established; in fact the orcokinin
receptor has yet to be identified. In this study, the presence of PSMs mapping to AST-B, FLPs,
SIFamide, myosuppressin, and orcokinins with sequence similarity to those previously found in other
crustacean species expands our understanding of the conservation of these NPs across species, and the
high degree of homology may indicate that these NP families are critical to neurotransmission and
neuroendocrine regulation in many organisms.
Other NPs observed in some of these species but not identified in Orconectes rusticus may still
be present. In addition to A-type ASTs, CHH, and MIH mentioned previously, C-type ASTs,
allatotropins, crustacean cardioactive peptide (CCAP), corazonin, kinins, YRamide, RYamides,
proctolin, pyrokinin, red pigment concentrating hormone (RPCH), and tachykinin-related peptides
have also been observed in decapod crustacean species. Some PSMs mapped to the known sequences
of these peptides or their preprohormones, but these matches represented portions of the sequence not
previously identified by MS/MS in these species, and they will be discussed in a separate section. It is
probable that the peptides not identified in this study but expected to be present in this species are 1)
found in neural organs other than the brain and eyestalk, 2) small peptides not well studied with
MS/MS interpretation software, 3) large peptides that must be processed enzymatically prior to
MS/MS analysis for identification, 4) present in very low quantities, and/or 5) subject to extensive
post-mortem degradation. Other ganglia typically analyzed by MS/MS for NP quantitation include the
thoracic and abdominal ganglia, the commissural organs, the stomatogastric ganglion, the pericardial
organ, and several large nerves. These were not analyzed in this study due to more extensive
dissection requirements. In addition, a multi-faceted mass spectrometric approach has been
demonstrated to provide more complete coverage of a species’ neuropeptidome [29, 31, 32, 38, 39, 42,
45-49]. Although several neuropeptide families were not found in this study, it is possible that they do
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exist in O. rusticus; other experimental approaches may be able to identify them. Variation in NP
expression profiles among species is also a possible explanation, and the absence of some NPs in
Orconectes rusticus may be important to its physiology or behavior. For instance, it is possible that
other less aggressive species may have NPs involved in social bonding, similar to oxytocin in
mammals. Not having an NP with this function may be one reason for increased aggressiveness in O.
rusticus.
5.4.2. Identification of PDH and CPRP Isoforms in Orconectes rusticus
Five isoforms of CPRP and 3 of PDH have been previously identified in the related crayfish
species Orconectes limosus [20], and the sequences of these peptides are compared to the PSMs
obtained in this study in Figures 5.4 and 5.5 and Table 5.2. Two groups of NPs that have high
sequence homology across many crustacean species are the CPRPs and PDHs. Due to the high degree
of sequence similarity and overlap between known crustacean CPRPs and PDHs, it is difficult to
determine exactly how many mature peptides could be generated from the obtained PSMs in this
study. PSMs for previously known NPs and novel products of the genes for these peptides were
observed in this study. The CPRPs observed all align to previously described Orconectes limosus
CPRPs A, A*, and B (Fig. 5.4.). These precursors could be responsible for all observed PSMs.
Extensive coverage of the CPRPs was achieved, and most sequences came from eyestalk extracts
(Table 5.2). This is expected, due to the localization of CHH primarily in the sinus gland of the
eyestalk. The absence of any mutated residues indicates that CPRPs are highly conserved between
these two Orconectes species. Comparison to known CPRP sequences from other crustaceans [20, 26,
29, 42, 47, 50-61] also suggests a great deal of homology. Sequence conservation throughout
evolutionary time may indicate that this peptide has an important biological function, instead of being
simply a byproduct of CHH synthesis. This study of Orconectes rusticus identified 31 PSMs,
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primarily from the sinus glands, that map to three distinct CPRPs that were previously identified in O.
limosus and that have a high degree of homology to other crustacean CPRPs.
Sequences for the pigment dispersing hormone (PDH) sequences are longer and more varied
between species, although they are also more commonly found in the eyestalk. Several PSMs were
made to peptides of the PDH family, as indicated in Fig. 5.5 and Table 5.2. In Fig. 5.5A, alignments
are made to the N-terminal portion of the peptide, which contains a precursor-related peptide only seen
by MS in a single previous study (Callinectes sapidus, pQELHVPEREA [29]). This portion of the
sequence has been characterized primarily by genetic techniques until now. The presence of these
PDH-precursor related peptides may indicate analysis of incompletely processed PDH, or may suggest
that these compounds are signaling molecules in their own right. The pyroglutamic acid residue at the
N-terminus and amidated C-termini are common PTMs seen in authentic NPs. Currently, only one full
PDH gene in Orconectes limosus has been sequenced, and several PSMs identified in this study do not
match with portions of this gene. Indeed, two PSMs match only to the PDH gene found in the
Cladoceran crustacean Daphnia magna. By parsimony, a minimum of 10 unique sequences would be
needed to cover PSMs that map to the N-terminal portions of PDHs. As indicated in Fig. 5.5B,
although only one PDH gene has been identified in Orconectes, multiple active PDH peptides have
been identified. These peptides may thus be products of an additional PDH gene or genes. Also shown
in Fig.5.5B, one PDH sequence must come from a non-Orconectes PDH sequence, PDH-I of
Marsupenaeus japonicus. This study mapped 17 PSMs to the N-terminus of PDH, but the currently
described Orconectes PDH gene is insufficient to describe the variety of sequences observed here. In
addition, two PSMs mapped to this portion of the PDH gene from the distantly related Daphnia
magna. Finally, 4 different C-terminal PDH sequences corresponding to the three previously known
O. limosus PDHs plus PDH-I from M. japonicus were observed with a total of 11 PSMs. This study
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has greatly increased the number of known PDH sequences and further suggests that the C-terminal
portion of the prepro-PDH gene may constitute a unique NP.
5.4.2. Neuropeptides Not Previously Identified by MS/MS in Crustaceans
An additional 47 PSMs were made to 43 unique identified neuropeptide sequences (some
PSMs mapped to the same identified peptide) (Table 5.3). These matches were made to compounds
previously only identified by genetic means, and often in the portions of the sequence not typically
associated with an active peptide. PSMs were found in the non-peptide regions of crustacean
cardioactive peptide (CCAP), FMRFamide-like peptides, red pigment concentrating hormone (RPCH),
orcokinin, SIFamide, and tachykinin. These may indicate new precursor-related peptide products of
the same genes responsible for producing those well-known neuropeptides. An alternative explanation
is that these fragments are left over after production of the genuine NP. Several PSMs mapped to
crustacean hyperglycemic hormone (CHH) fragments, although too few were obtained to determine a
partial sequence for this peptide in O. rusticus. Additional CPRP isoforms could be suggested by
several matches to CPRPs other than those previously found in O. limosus. Matches to bursicon-α
were found for the first time using MS/MS (alignment in Fig. 5.6). The presence of this peptide in
crustacean genomes has previously been noted, but had not been found using MS/MS techniques [26,
47]. Four of these matches appear in the signal peptide region, which would presumably be cleaved
post-translationally. Although the coverage of the bursicon-α gene is not high, these results are
encouraging for further analysis, particularly targeted bottom-up proteomics approaches. Two
overlapping PSMs were found for a calcitonin-like diuretic hormone (CLDH) from Homarus
americanus [62]. They map to a region of the gene immediately preceding the putative mature peptide,
which could create a precursor-related peptide. Other matches include crustacean female hormone,
eclosion hormone, eyestalk peptide, an insulin-like peptide, and an oxytocin-neurophysin-like peptide.
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Many of these peptides have multiple PSMs from multiple organs and/or analysis techniques, thus
increasing the confidence of their assignments. These matches are not sufficient to positively state the
presence of these peptides in Orconectes rusticus¸ mostly because they do not provide full coverage of
sequences of the hormones in question. Many of these are large hormones that would need additional
treatment prior to MS/MS analysis to map their sequences. These additional PSMs are intriguing and
suggest that additional precursor-related peptides may be encoded by the same genes that encode
known NPs, and that several NPs not previously detected with MS/MS may be present in O. rusticus.
5.4.3. Comparison of Data Sets
Results for PSMs mapping to proteins or unidentified transcripts of the Daphnia pulex genome
are presented in Appendix C. The D. pulex genome has already been subjected to genome mining for
neuropeptide sequences [63, 64], but it is possible that some of these still unidentified proteins produce
NPs that were missed in the previous studies. A number of spectra were assigned sequences by de
novo sequencing only with PEAKS. The identities of these were interrogated by BLAST, and it was
determined that no neuropeptides or hormones were found in this data set, although several PSMs
mapped to unidentified D. pulex proteins, which may yet contain undiscovered NP sequences. This list
is also included in Appendix C.
Both software programs performed equally in terms of numbers of identifications in both brain
and eyestalk (Fig. 5.7.). The number of PSMs made in eyestalk, however, was much greater than in the
brain (Fig. 5.8.), and very few NPs overlapped. This is likely due to the presence of large NPs in the
eyestalk, such as CPRPs and PDHs. Smaller NPs typically found in the brain, such as ASTs and FLPs,
were not identified well due to problems mentioned earlier. Many NPs have a tissue-specific pattern of
distribution. Interestingly, only CPRPs and PDHs had a clear family trend in tissue distribution—most
were present in the eyestalk. It is likely that the addition of these two groups to the eyestalk tally led to
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the overall identification of more NP sequences in this tissue, as CPRPs and PDHs had many PSMs.
The distribution of PSMs to neuropeptide families is indicated in Fig. 5.9.
As mentioned previously, each PSM may indicate a unique neuropeptide with biological
activity, or they may map to fragments of authentic NPs. For this reason, the PSMs were assembled
into the lowest number of possible NPs that could produce the observed peptides. The least possible
number of NP precursors that could explain the NPs observed to match the 8 NP families for which
identifications were made to previously observed compounds was determined by parsimony and the
resulting peptides are indicated in Table 5.4. Twenty-three NPs are sufficient to explain these PSMs,
although it is expected that this number is a very low estimation.
5.5. Conclusions and Future Work
In this work, the neuropeptides present in the crayfish Orconectes rusticus were catalogued
using MS/MS. This species is of particular interest to neuroscience, for the behavioral studies
conducted in it, and ecology, for its impact on the environment as an invasive species. This list of NPs
will be of aid for further studies into this organism, which will be useful in understanding how an
organism responds to changing environmental conditions via neuroendocrine regulation, among other
things. Two MS/MS interpretation approaches were used: database search using the program Mascot
and de novo sequencing using the program PEAKS. Determining a score cutoff was difficult, as the
algorithms that these programs use to score the probability of a match are not optimal for small
peptides, as many NPs are, and small databases, such as the ones employed in this study. The mixture
of large preprohormones and small active NPs in the combination database used may also have
contributed to difficulties obtaining scores that accurately represent the quality of a PSM. It is
suggested that a different set of criteria be used for databases containing only short sequences of active
NPs than for protein/preprohormone databases. A lower score threshold, or even a different scoring
229
mechanism could be used to ensure that all NPs present are fairly assigned. Lowering the score
threshold will increase the manual work, as spectra will have to be verified by hand, but development
of a new algorithm to score short peptide matches to spectra requires expertise in statistical methods.
This study identified at minimum one B-type allatostatin, 3 CPRPs, 2 FMRFamide-like
peptides, 1 myosuppressin, 8 orcokinins, 1 orcomyotropin, 6 PDHs, and one SIFamide from
previously identified NPs in the extracts of brain and eyestalk. Potentially novel PDHs were
discovered, which may represent a new class of PDH-precursor related signaling molecules, which
were first suggested by Hui and colleagues [29]. A total of 98 peptide-spectrum matches were made to
NPs previously observed in other decapod crustacean species. PSMs for signaling molecules not
previously identified in decapod crustaceans were also made. Other PSMs map to previously
unobserved (by MS) sequence regions of CHHs, bursicon, FMRFamides, a crustacean female
hormone, insulin-like hormones, calcitonin-like diuretic hormones (CLDHs), eclosion hormone,
eyestalk peptides, an oxytocin-neurophysin-like hormone, red pigment concentrating hormone
(RPCH), and tachykinins. These peptides warrant further study, as many have homology to
mammalian NPs involved in critical physiology.
5.6. Respective Contributions Statement and Acknowledgments
CMS designed and carried out the research and wrote the paper. ZL aided in sample
acquisition and preparation. LL advised and revised the paper.
Nicole Woodards and Weifeng Cao of the Li Lab are acknowledged for assisting in database
preparation. Alex Latzka of the Vander Zanden Lab and coworkers at the Trout Lake Research
Station, Department of Limnology are acknowledged for crayfish collection. The School of Pharmacy
Analytical Instrumentation Center is acknowledged for access to the Mascot software.
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233
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5.8. Figures
Figure 5.1. Reproducibility of PSMs in A) Mascot and B) PEAKS. Incomplete overlap is likely a
result of instrumental variability in the selection of precursors for MS/MS, although it could be due to
biological variation and false-positive PSMs.
A) Mascot
Eyestalk 1
33
Both
19
Eyestalk 2
17
B)PEAKS
Eyestalk 1
36
Brain 1
9
Both
11
Brain
2
12
P
Both
15
Eyestalk 2
27
Brain 1
21
Both
9
Brain 2
16
Figure 5.2. Comparison of peptide spectrum matches (PSMs) from Mascot and PEAKS. A) A PSM
obtained with Mascot with a score of 18 that shows a poor degree of peak-fragment correlation. B) A
PSM obtained with Mascot with a score of 15 showing good correlation between MS/MS peaks and
peptide fragments. C) A PSM from PEAKS with a score of 15.07 that matches poorly. D) A PSM
from PEAKS with a score of 16.85 that matches well. Some additional high-intensity peaks without
assignments map to neutral losses
234
A)
B)
C)
D)
235
Figure 5.3. Alignment of previously identified orcokinin sequences. PSMs for orcokinin peptides that
have previously been observed via MS/MS in decapods are aligned. Peptides are marked with a “u” in
the Unique column cannot be explained as fragments of the other peptides. By parsimony, these 16
PSMs can thus be mapped to 8 unique peptides.
PSM
DFDEIDRSGFA
NFDEIDR
NFDEIDRS
NFDEIDRSGFA
NFDEIDRSGFG
NFDEIDRSGFGF
NFDEIDRSGFGFA
NFDEIDRSGFGFN
FDEIDRSGFGFN
EIDRSGFGFN
NFDEIDRSGFGFV
NFDEIDRSSFG
NFDEIDRTGFamide
NFDEIDRTGFG
NFDEIDRTGFGF
NFDEIDRTGFGFH
Unique
u
u
u
u
u
u
u
u
236
Figure 5.4. Alignment of CPRP PSMs. A lowercase m indicates an oxidized methionine. Orconectes
limosus CPRP sequences are taken from reference and compared to sequences obtained in this study.
Red indicates the residue that distinguishes CPRP A* from A, and green indicates the residue that
distinguishes B from the others. The column to the far right shows which CPRP each fragment maps
to. For those fragments that occur in multiple CPRPs, it is left blank.
Olim CPRP A
Olim CPRP A*
Olim CPRP B
RSVEGSSRMERLLSSGSSSSEPLSFLSQDHSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRmERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVNa mide
RSVEGSSRMERL
RSVEGSSRMERLL
GSSRMERLL
MERLLSSGSSSSEPLSFLSQDQSVS
RLLSSGSSSSEPLSFLSQDQSVS
RLLSSGSSSSEPLSFL
RLLSSGSSSSEPLSF
RLLSSGSSSSEPL
RLLSSGSSSSEPLS
LLSSGSSSSEPLS
LLSSGSSSSEPLSF
LLSSGSSSSEPLSFL
LLSSGSSSSEPLSFLSQ
LLSSGSSSSEPLSFLSQDQSVS
LLSSGSSSSEPLSFLSQDQSVN
LSSGSSSSEPLSFL
LSSGSSSSEPLSFLSQDQSVS
SSGSSSSEPLSFLSQDHSV
SSGSSSSEPLSFLSQDQSVS
SGSSSSEPLSFL
SGSSSSEPLSFLSQDQSVS
SGSSSSEPLSFLSQDQSVN
SSSSEPLSFLSQDQSVS
SSSEPLSFLSQDQSVS
SSEPLSFLSQDQSVS
SEPLSFLSQDQSVS
PLSFLSQDQSVS
CPRP
CPRP
CPRP
CPRP
A*
A*
B
B
CPRP A*
CPRP A*
CPRP A*
CPRP B
CPRP A*
CPRP A
CPRP A*
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
A*
B
A*
A*
A*
A*
A*
237
Figure 5.5. Alignment of PDH sequences. A lowercase m indicates an oxidized methionine. A
lowercase l in a PSM sequence indicates a L or I residue, since they cannot be distinguished using MS.
A lowercase p in front of a Q indicates the pyroglutamic acid form of Q. A) Alignment of sequences to
the N-terminal portion of the precursor. Active peptides formed from this portion are sometimes called
PDH precursor-related peptides. Reference sequences are given first, followed by several rows of
PSMs from the data. Underlined sequences differ from the literature sequence for O. limosus. Peptides
that are unique, in that they cannot be contained in any other PSM, are indicated with a letter u in the
right-most column. In the Daphnia magna sequence, the peptides that are matched are highlighted in
bold. B) Alignment to the C-terminal portion. This is the active PDH peptide. Sequences for PDH A,
B, and C from O. limosus are indicated as Olim PDH-A, -B, -C. Sequence for Marsupenaeus
japonicus PDH I is indicated as Marj PDH-I. Red indicates residues that differ between Olim PDH-A
and –B. Green indicates the residue that differs between Olim PDH-B and C. Blue indicates the
residues that differ between Olim PDH-A and Marj PDH-I. PSMs follow the reference sequences,
with modified residues indicated by colors, and the precursor that PSM requires indicated in the
righthand column. For PSMs where multiple precursors are suggested, this column is left blank.
238
A. N-Terminus
Orconectes limosus PDH precursor (S59496.1) 1-56
MRSAMVVLVLVAMVAVFTRAQE LKYPEREVVAELAAQIYGWPGSLGTMAGGPHKRNSELINSILGLPKVMNEAGRR
MVAmSlQlT
u
pQElKYPEREVVAEl
pQElKYPEREVVAElA
pQElKYPEREVVAElAA
pQElKYPEREVVAElAAQ
u
SSlKYFEREVVSElAAQll
SSlKYFEREVVSElAAQllR
SSlKYFEREVVSElAAQllRV
u
EAVATlAAHllKVVCAPlEGAGGlPHKamide
u
VV lAAVlTQG
u
AAQllRVAQGPSAFVAGPHK
u
VAQGPSAFVAGPHK
AQGPSAFVAGPHK
lGTmAGGPHKamide
u
AGGlPHKRNamide
u
EYllKFamide
u
DlNPTEKamide
u
Daphnia magna PDH precursor (CAA72409.1)
MHQLSAKLSHFSIALLVLFVSLTMDVESAPAATSSINRPEAQLSIQEMEKFLEGLTRYLHRQRLEQQRMH
QPQAKSLMSEEVTYDPDAIDRAILGEMAAPLDAERSSSVELANNSMSHARPPTNNKWPWSLSNFERIEDQ NVKQRQPYGK
SlTmDVES
SlmSEEVamide
B. C-Terminus
Olim PDH-A NSELINSILGLPKVMNEAamide
Olim PDH-B NSELINAILGSPTLFGEVamide
Olim PDH-C NSELINAILGSPTLMGEVamide
Marj PDH-I NSELINSLLGIPKVMTDAamide
NSEllNSllGlPKVMNEAamide
NSEllNSllGl
NSEllNSllGlP
NSEllNSllGlPK
SEllNSllGlPamide
SllGlPKVm
SllGlPKVMNEAa
llGlPKVMNEAa
llGlPKVmT
NSEllNAllGSPTlM
NSEllNAllGSPTl
Olim PDH-A
Marj PDH-I
Olim PDH-C
239
Figure 5.6. Alignment of PSMs with bursicon alpha sequences. PSMs are indicated by color with
where they align on the bursicon alpha gene. Two overlapping PSMs are both indicated with red, and a
third with some overlap is indicated in blue. Where those overlap, the sequence is colored purple. The
underlined portion of the gene sequences is the signal peptide. A lowercase M indicates an oxidized
methionine.
Bα-Cs ACG50067.1 bursicon hormone alpha subunit [Callinectes sapidus]
Bα-Cm ABX55995.1 bursicon alpha [Carcinus maenas]
Bα-Hg ADI86242.1 bursicon alpha subunit [Homarus gammarus]
Bα-Cs MNSNLTWAMVGAAVTVLVVIGVDVARADECSLRPVIHILSYPGCTSKPIPSFACQGRCTSYVQVSGSKLW
Bα-Cm MMSSLPWTVVGAAVTVLVVIGVGVAQADECSLRPVIHILSYPGCTSKPIPSFACQGRCTSYVQVSGSKLW
Bα-Hg
MGGLSWVLMVLGVATVVWSDECSLTPVIHILSYPGCVSKPIPSFACQGRCTSYVQVSGSKLW
AmVGAAVT
mVGAAVTV
VTVLVVIG
TVVWSDECSLT
Bα-Cs QTERSCMCCQESGEREAAITLNCPKPRPGEPKEKKVLTRAPIDCMCRPCTDVEEGTVLAQEIANFIQDSP
Bα-Cm QTERSCMCCQESGEREAAITLNCPKPRPGEPKEKKVLTRAPIDCMCRPCTDVEEGTVLAQKIANFIQDSP
Bα-Hg QTERSCMCCQESGEREASVVLNCPKVRKGEPTRRKILTRAPIDCMCRPCTDVEEGTVLAQEIANFIHDSP
VLTRAPIDCMCRPCT
Bα-Cs MDSVPFLK
Bα-Cm MDSVPFLK
Bα-Hg MGNVPFLK
Figure 5.7. Overlap of the two software programs in PSM identifications.
B. Brain
A. Eyestalk
PEAKS
28
Both
45
Mascot
23
PEAKS
26
Both
20
Mascot
12
240
Figure 5.8. Overlap in identifications between tissues.
Mascot
PEAKS
Eyestalk
65
Both
8
Eyestalk
64
Brain
38
Both
4
Brain
28
Figure 5.9. PSMs mapped to each NP family in the brain and eyestalk tissues. Abbreviations in text.
Total PSMs in eyestalk: 96. Total PSMs in brain: 58.
Eyestalk
Brain
CPRP
PDH
Orcokinin
CHH
SIFamide
Bursicon
FMRFamide
Myosuppressin
AST-B
Crustacean female hormone
Insulin-like
Orcomyotropin
Calcitonin
CCAP
EH
Eyestalk peptide
Oxytocin-neurophysin
RPCH
Tachykinin
Orcomyotropin
SIFamide
FDAFTTGFGHS
GYRKPPFNGSIFa
YAIAGRPRFamide
pQDLDHVFLRFamide
FMRFamide
Myosuppressin
Orcokinin
TNWNKFQGSWamide
EPLDHVFLRFamide
NP Matching Sequence
AST-B
FMRFamide
NP Family
and Mascot scores are both -10logP.
TNWNKFQGSWa
EPLDHVFLR
EPLDHVFLRF
EPLDHVFLRFa
YAIAGRPR
pQDLDHVFLR
pQDLDHVFLRF
pQDLDHVFLRFa
DFDEIDRSGFA
EIDRSGFGFN
FDEIDRSGFGFN
NFDEIDR
NFDEIDRS
NFDEIDRSGFA
NFDEIDRSGFG
NFDEIDRSGFGF
NFDEIDRSGFGFA
NFDEIDRSGFGFN
NFDEIDRSGFGFV
NFDEIDRSSFG
NFDEIDRTGFa
NFDEIDRTGFG
NFDEIDRTGFGF
NFDEIDRTGFGFH
FDAFTTGFGHS
GYRKPPFN
Sequence of PSM
Eyestalk
Brain
PEAKS Score Mascot Score PEAKS Score Mascot Score
32.36
35.44
16.55
51.99
32.87
33.24
35.44
29.98
16.55
53.35
48.09
29.92
45.87
47.52
30.71
20.73
19.91
18.8
23.52
33.66
22.13
49.52
51.82
46.25
58.31
75.5
46.55
53.36
41.7
48.84
45.69
46.01
76.44
101.5
75.71
64.87
69.98
58.89
16.85
21.59
28.28
36.02
37.43
34.47
27.52
21.27
64.5
63.15
38.98
43.02
19.25
19.96
Table 5.1. PSMs that map to putative neuropeptides previously identified in decapod crustaceans. Abbreviations in text. PEAKS
241
GYRKPPFNG
GYRKPPFNGSIF
GYRKPPFNGSIFa
KPPFNGSIF
KPPFNGSIFa
RKPPF
RKPPFN
RKPPFNG
RKPPFNGSI
RKPPFNGSIF
RKPPFNGSIFa
49.1
53.8
36.15
17.94
27.45
21.18
23.42
30.71
56.9
64.97
21.04
20.16
30.91
39.46
38.42
23.7
33.66
21.41
17.23
242
NP
Family
CPRP
GSSRMERLL
LLSSGSSSSEPLS
LLSSGSSSSEPLSF
LLSSGSSSSEPLSFL
LLSSGSSSSEPLSFLSQ
LLSSGSSSSEPLSFLSQDQSVN
LLSSGSSSSEPLSFLSQDQSVS
LSSGSSSSEPLSFL
LSSGSSSSEPLSFLSQDQSVS
MERLLSSGSSSSEPLSFLSQDQSVS
PLSFLSQDQSVS
RLLSSGSSSSEPL
RLLSSGSSSSEPLS
RLLSSGSSSSEPLSF
RLLSSGSSSSEPLSFL
RLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRM(O)ERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRMERL
RSVEGSSRMERLL
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVNamide
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
SEPLSFLSQDQSVS
SGSSSSEPLSFL
SGSSSSEPLSFLSQDQSVN
SGSSSSEPLSFLSQDQSVS
SSEPLSFLSQDQSVS
Sequence of PSM
Eyestalk
PEAKS Score Mascot Score
16.43
25.83
33.61
43.97
24.56
64.89
34.29
51.49
58.65
83.14
51.41
109.36
110.22
21.4
45.31
33.81
116.9
151.69
64.49
82.22
40.65
46.53
63.06
94.94
70.99
92.77
54.37
59.16
92.46
100.88
47.02
28.97
15.38
81.38
71.45
129.83
38.04
16.35
21.71
24.69
87.6
73.48
28.58
20.08
Brain
PEAKS Score Mascot Score
Lowercase l indicates either a I or L residue, as these are isobaric. PEAKS and Mascot scores are both -10logP.
Table 5.2. PSMs for CPRP and PDH. Abbreviations in text. M(O) indicates an oxidized methionine. pQ indicates pyroglutamate.
243
PDH
SSGSSSSEPLSFLSQDHSV
SSGSSSSEPLSFLSQDQSVS
SSSEPLSFLSQDQSVS
SSSSEPLSFLSQDQSVS
AAQllRVAQGPSAFVAGPHK
AGGlPHKRNamide
AQGPSAFVAGPHK
DlNPTEKamide
EAVATlAAHllKVVCAPlEGAGGlPHKamide
EYllKFamide
lGTM(O)AGGPHKamide
llGlPKVM(O)T
llGlPKVMNEAamide
MVAM(O)SlQlT
NSEllNAllGSPTl
NSEllNAllGSPTlM
NSEllNSllGl
NSEllNSllGlP
NSEllNSllGlPK
NSEllNSllGlPKVMNEAamide
pQElKYPEREVVAEl
pQElKYPEREVVAElA
pQElKYPEREVVAElAA
pQElKYPEREVVAElAAQ
SEllNSllGlPamide
SllGlPKVM(O)
SllGlPKVMNEAamide
SlM(O)SEEVamide
SlTM(O)DVES
SSlKYFEREVVSElAAQll
SSlKYFEREVVSElAAQllR
SSlKYFEREVVSElAAQllRV
VAQGPSAFVAGPHK
28.04
18.82
30.95
15.39
34.24
24.73
33.91
25.29
31.32
25.71
38.13
41.45
65.89
76.48
102.29
19.06
19.79
48.58
36.65
69.16
45.91
21.47
32.27
50.76
82.63
67
16.23
16.66
26.61
29.27
24.71
34.91
86.67
35.58
38.6
15.74
19.11
47.45
21.08
55.2
51.55
17
21.09
16.58
29.83
19.03
36.46
37.37
16.36
25.71
244
19.05
Peptide Family
Bursicon
Bursicon
Bursicon
Bursicon
Bursicon
Calcitonin
Calcitonin
CCAP
CCAP
CCAP
CHH
CHH
CHH
CHH
Accession
Number
ABX55995.1
ACG50067.1
ACG50067.1
ACG50067.1
ADI86242.1
ACX46386.1
ACX46386.1
ABB46292.1
BAF34909.1
BAF34909.1
AAD45236.1
ACN65120.1
ACS35346.1
AER27833.1
Bursicon hormone alpha subunit
[Callinectes sapidus]
Bursicon hormone alpha subunit
[Callinectes sapidus]
Bursicon hormone alpha subunit
[Callinectes sapidus]
Bursicon alpha subunit [Homarus
gammarus]
Prepro-calcitonin-like diuretic
hormone [Homarus americanus]
Prepro-calcitonin-like diuretic
hormone [Homarus americanus]
Crustacean cardioactive peptide
[Homarus gammarus]
Crustacean cardioactive peptide
[Procambarus clarkii]
Crustacean cardioactive peptide
[Procambarus clarkii]
Hyperglycemic hormone-like
[Metapenaeus ensis]
Crustacean hyperglycemic hormone
[Panulirus homarus]
Crustacean hyperglycemic hormone
isoform 1 [Rimicaris kairei]
Crustacean hyperglycaemic hormone
[Ptychognathus pusillus]
Bursicon alpha [Carcinus maenas]
Description
starting with LL are from the in-house database LiDB.
PAGHPLEKRQamide
HPSGLAALTASH
SPPPAGRTATVIGCSVNVSTTYamide
pEAASPGASSPWVEHR
KLWEQLQ
AGPLAKRDIG
TPHTQPR
TRLGHSIIRANELEKF
LTRLGHSIIRANELEKFVRSSGSA
TVVWSDECSLT
VLTRAPIDCMCRPCT
M(O)VGAAVTV
AM(O)VGAAVT
VTVLVVIG
PSM Sequence
15.04
22.2
17.91
15.02
19.46
19.38
21.51
15.38
18.99
PEAKS
PEAKS
37.72
Brain
Eyestalk
Mascot
18.17
18.3
58.81
28.86
Mascot
known NP genes. M(O) indicates an oxidized methionine. Pyroglutamate residues are indicated as pE or pQ. Accession numbers
Table 5.3. PSMs for NPs and hormones not previously identified with MS/MS in decapods. Some map to precursor regions of
VVlAAVlTQG
245
CHH
CHH
CHH
CHH
CHH
CHH
CPRP
CPRP
CPRP
CPRP
Crustacean
female hormone
Crustacean
female hormone
Crustacean
female hormone
EH
Eyestalk
peptide
Eyestalk
peptide
FMRFamide
FMRFamide
Insulin-like
AFD28272.1
AFG16934.1
AFM29133.1
LL206
O15981.1
O97384.1
AAM21927.1
AAS46643.1
ABS01332.1
LL343
ADO00266.1
AF112986_1
EFX80896.1
ADA67878.1
BAE06263.1
AF112986_1
AFK81936.1
ADO00266.1
ADO00266.1
CHH
AFD28272.1
FLRFamide precursor protein B
[Procambarus clarkii]
Putative sulfakinin-like peptide
[Daphnia pulex]
Insulin-like androgenic gland
hormone precursor [Penaeus
Eyestalk peptide [Jasus edwardsii]
Hyperglycemic hormone [Pandalopsis
japonica]
Crustacean hyperglycmic hormone
[Portunus pelagicus]
Crustacean hyperglycemic hormone
Hoa-CHH-A (pCHH-A[pQ61V132amide]) [Homarus amerianus]
Crustacean hyperglycemic hormone 5;
Pej-SGP-V [Marsupenaeus japonicus]
Crustacean hyperglycemic hormone 2;
Pm-SGP-II [Penaeus monodon]
Crustacean hyperglycemic hormone
precursor [Pachygrapsus marmoratus]
Crustacean hyperglycemic hormone
precursor [Macrobrachium
rosenbergii]
Prepro-crustacean hyperglycemic
hormone [Galathea strigosa]
CHH precursor-related peptide Capr
CPRP II [13-38] [Cancer productus]
Crustacean female hormone
[Callinectes sapidus]
Crustacean female hormone
[Callinectes sapidus]
Crustacean female hormone
[Callinectes sapidus]
Eclosion hormone [Amphibalanus
amphitrite]
Eyestalk peptide [Jasus edwardsii]
CHH protein [Scylla paramamosain]
CHH protein [Scylla paramamosain]
VGMLMVLSLT
PVELGSSNPamide
PAEYSRNVR
TPARQR
DVRQDGQTNamide
PGLASLGGSTST
QGPLPIQPVamide
ERQIQGP
DGGYIDLSENamide
GDLPGGLVHamide
LSFMPEHP
RIEKLLST
DRSLFGKL
SSPVASLIRG
LVAGASSAGT
LDDLLLSDV
APM(O)QGYGTEamide
EEHVDIR
pEGAAHPLEKRamide
LVACIAMATLPQTQamide
20.02
20.84
17.36
15.07
21.73
15.63
19.2
21.37
30.15
28.32
16.77
18.55
17.93
15.44
19.84
18.19
19.29
16.15
18.05
22.81
246
Insulin-like
Insulin-like
Orcokinin
Orcokinin
Orcokinin
Oxytocinneurophysin
RPCH
RPCH
SIFamiderelated peptide
Tachykinin
Tachykinin
Tachykinin
EFX76240.1
EFX76240.1
Q9NL83.1
Q9NL83.1
Q9NL83.2
ADD24046.1
ADQ73633.1
Q867W1.1
BAC82426.1
BAC82426.1
BAC82426.1
ADN95181.1
Insulin-like
EFX70023.1
Insulin/IGF/relaxin-like peptide 2
[Daphnia pulex]
Insulin/IGF/relaxin-like peptide 1
[Daphnia pulex]
Insulin/IGF/relaxin-like peptide 1
[Daphnia pulex]
Orcomyotropin/orcokinin precursorlike peptide [Procambarus clarkii]
Orcomyotropin/orcokinin precursorlike peptide [Procambarus clarkii]
Orcomyotropin/orcokinin precursorlike peptide [Procambarus clarkii]
Oxytocin-neurophysin 1
[Lepeophtheirus salmonis]
Red pigment concentrating hormone
precursor [Scylla paramamosain]
Red pigment concentrating hormone
precursor [Scylla olivacea]
GYRKPPFNGSIF-amide;
FRP1_PROCL [Procambarus clarkii]
Preprotachykinin [Procambarus
clarkii]
Preprotachykinin [Procambarus
clarkii]
Preprotachykinin [Procambarus
clarkii]
monodon]
HFDDESEIDAYIQAL
HFDDESEIDAY
AGMDSELETLL
AGGDSLYEPGK
PPGSSSGDSCGP
SVGGAPGGVVPSSPGSSSGDSamide
QYKQCSSCGP
VYVPRYIANLY
VYVPRYIAN
MTAQM(O)FTIALLLSLS
VTPAEP
M(O)IIPSTVGRCWM(O)
SLGLTTTAATPNamide
51.8
17.89
77.24
38.47
38.23
18.39
21.23
45.72
22.85
18.49
18.54
18.11
87.4
37.25
16.8
45.89
29.9
22.28
22.03
247
2
1
8
1
6
Myosuppressin
Orcokinin
Orcomyotropin
PDH
Least PossibleLowest
Number of Identified
NPs that Can Explain
Observed PSMs
1
3
FMRFamide-like peptides
AST-B
CPRP
NP Family
TNWNKFQGSWamide
RSVEGSSRMERLLSSGSSSSEPLSFLSQDHSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
EPLDHVFLRFamide
YAIAGRPR
pQDLDHVFLRFamide
DFDEIDRSGFA
NFDEIDRSGFA
NFDEIDRSGFGFA
NFDEIDRSGFGFN
NFDEIDRSGFGFV
NFDEIDRSSFG
NFDEIDRTGFamide
NFDEIDRTGFGFH
FDAFTTGFGHS
MVAmSlQlT
pQElKYPEREVVAElAAQ
SSlKYFEREVVSElAAQllRV
EAVATlAAHllKVVCAPlEGAGGlPHKamide
VVlAAVlTQG
Sequences
methionine. Lowercase l’s indicate either I or L residues. Pyroglutamic acid is indicated as pQ or pE.
required due to sequence variations being present at different parts of the overall sequence. A lowercase m indicates an oxidized
Table 5.4. NPs required to explain all observed PSMs, and sequences of unique PSMs. There are more unique sequences than NPs are
248
SIFamide
1
AAQllRVAQGPSAFVAGPHK
lGTmAGGPHKamide
AGGlPHKRNamide
EYllKFamide
DlNPTEKamide
SlTmDVES
SlmSEEVamide
NSELINSILGLPKVMNEAamide
NSELINAILGSPTLFGEVamide
NSELINAILGSPTLMGEVamide
NSELINSLLGIPKVMTDAamide
GYRKPPFNGSIFamide
249
250
Chapter 6: Development of Methods for Neuropeptide Quantitation with DataIndependent MS/MS
Modified from Schmerberg, C. M. and Li, L. Rapid Commun. Mass Spec. [in preparation]
6.1. Abstract
Quantitation using mass spectrometry (MS) and tandem MS (MS/MS) is not always
straightforward due to a number of factors, most notably run-to-run variability and variability of
some aspects of MS (primarily in ionization efficiency) based on molecular properties.
Specialized methods for highly accurate quantitation using MS and MS/MS have been
developed, but these typically require isotopic labeling and/or prior method optimization for the
analytes of interest. Therefore, most experimental strategies for samples of unknown
composition employ two steps and two separate instrument runs, one for identification of the
components of the sample, followed by one for quantification of these identified components [1].
A new method of MS/MS data acquisition, called data-independent acquisition (DIA), has been
developed to allow simultaneous identification and quantification of unknown samples. This
method differs from typical MS/MS in that it fragments all ions concurrently, instead of selecting
single ions for fragmentation. The precursor and fragment ions can be aligned to each other by
tracking their retention times, charge states, and masses. The fragment ions can be quantified,
which yields quantitative information about their precursors, in a scheme similar to selectedreaction-monitoring (SRM) methods [2]. In this chapter, an open-source, vendor-neutral software
designed for SRM quantitation (Skyline) [3] is applied to highly multiplex reaction monitoring
(all reactions monitoring, ARM) quantitation of neuropeptides (NPs) using DIA MS/MS data.
This is the first work demonstrating use of this software for DIA MS/MS quantitation, and the
first to quantify endogenous peptides from DIA data as opposed to larger proteins with multiple
251
peptide fragments. After establishment of the quantitation method, proof-of-principle was
established by quantifying NPs in neuroendocrine tissues of the blue crab Callinectes sapidus
after feeding.
6.2. Introduction
Tandem mass spectrometry (MS/MS) is a highly useful analytical tool both for
identification of molecules and their quantification. However, it typically cannot conduct both
identification and quantification simultaneously, because different mass spectrometer (MS)
parameters are typically required for each type of experiment. Notable exceptions exist, such as
spectral counting and tandem mass tags, but those methods have disadvantages that will be
detailed in following sections. There are several reasons different parameters would be required
for identification and quantification, but unifying feature is that mass filters are typically used for
both approaches, albeit in different ways. These mass filters either prevent monitoring multiple
fragments in MRM-type experiments, which is required for identification, or prevent enough
measurements of fragment ion intensities to be obtained across a given peak (i.e. too few data
points/peak), which is required for quantitation. Data-independent analysis (DIA) MS employs
no mass filter but produces both precursor and fragment ions. This increases the complexity of
data interpretation, but it is possible to quantitate the product ions of specific precursors in a
pseudo-multiple reaction monitoring (pMRM), or all-reactions monitoring (ARM), manner, as
well as to determine identity in a single run. For further information in MS-based quantitation, a
review is available [1].
6.2.1. Methods for Quantitation by MS/MS
The “gold standard” of quantitative MS/MS, in terms both of quantitation accuracy and
sensitivity, is selected-reaction-monitoring (SRM). This is typically conducted in a liquid
252
chromatography-triple quadrupole mass spectrometer setup. The robustness of SRM lies in that it
is essentially counting the number of ions with a given precursor mass and a given fragment
mass that travel through the instrument to the detector, by using the first and last quadrupoles as
mass filters. The first quadrupole permits only the precursor molecule to enter the second
quadrupole, where collision/fragmentation occurs, and the third quadrupole permits only a given
fragment produced by that fragmentation reaction to pass through to the detector (See Fig. 6.1).
Multiple compounds can be quantified by cycling the quadrupoles through a given set of
precursor and fragment masses over time, which is called multiple reaction monitoring (MRM),
and if the retention times of analytes of interest are also known, the instrument can carry out a
scheduled SRM at that time, as another method to permit multiplexed quantitation. The behavior
of a given fragment during and between runs in this environment is highly reproducible, and the
number of transitions monitored at any given time is limited so that enough data can be collected
to obtain a Gaussian peak profile (usually >6 points), when ion intensity is plotted against time.
The intensity of the analyte(s) of interest is usually tracked against that of an internal standard,
for relative or absolute (with calibration curves) quantitation. A vendor-neutral, open-source
software for MRM experiments has been developed by researchers at the University of
Washington (Skyline) [3].
6.2.2. Methods for Identification by MS/MS
In identification, the selection of precursors for fragmentation is typically carried out in a
scheme called data-dependent acquisition (DDA). MS1 scans are conducted, permitting all
analytes to go to the detector, and from that data, several high-intensity precursors are selected
for fragmentation. Each precursor is then sequentially selected by the first mass analyzer and
fragmented, followed by detection of all of its fragment ions. The number of precursors that will
253
be monitored is dependent on the experimental parameters and the speed and resolution of the
instrument. Using a high resolution time-of-flight (TOF) analyzer for the MS2 scan, only a
handful of precursors can be monitored at a time because the separation of ions is relatively slow
compared to the speed at which precursors elute from an ultrahigh performance liquid
chromatography (UPLC) column, typically used prior to MS ionization. The MS2 spectrum that
results and its associated precursor can be interpreted to determine the identity of that precursor.
Typically, only high-intensity ions are selected as precursors, to provide higher-quality MS2
spectra. In addition, only a few scans of each precursor-fragment ion combination are acquired,
because with high-quality MS2 spectra, only a few spectra are required to provide sequence
information. Therefore, not enough data points are obtained for SRM-style quantitation.
Occasionally, the intensity of an ion in the MS1 spectrum over time can be determined from
DDA MS/MS experiments, but that is only possible with faster-scanning lower-resolution
instruments such as ion traps, and the specificity is reduced compared to MS2 ion monitoring.
This can be combined with MS1-level quantification strategies including isotope-coded affinity
tags (ICAT), chemical modification with isotopic reagents, and incorporation of heavy amino
acids, as well as label-free quantitation employing internal standards [1]. Although these can be
used for quantitation based on the MS1 scan concurrent with identification based on MS2 scans,
the quantitation is not as accurate.
6.2.3. Methods for Simultaneous Quantitation and Identification by MS/MS
Alternative methods exist for identification and quantitation in a single run. For MS/MS
experiments with high scan rates, the number of times an ion is selected for MS/MS analysis can
be used as a measure of its abundance, called its spectral count [1]. This method is most robust
for quantification at the protein level based on multiple peptides identified, and is not applicable
254
for endogenous neuropeptide (NP) study. A NP-compatible approach employs isotopic MS2
fragment ions produced by tags that are isobaric at the MS1 level. One example of this is DiLeu
labels developed by the Li laboratory [4]. These tags use isotopes to generate characteristic
fragment ions of given masses, and the relative intensities of these ions can be used for
quantification. Finally, several transitions can be monitored with full fragment scans in some
high-speed instruments, allowing for simultaneous identification and quantification of those
predefined transitions with Skyline SRM software [5]. Although several methods for
simultaneous quantification and identification exist, they are not optimal for experiments
attempting to discover and quantify endogenous peptides, such as NPs.
A new method of MS data acquisition, termed data-independent acquisition (DIA), has
been developed for simultaneous identification and quantification of molecules (Fig. 6.2). This
method is fundamentally different from other MS/MS methods in that mass filtering is not
employed, either at the MS1 level as in DDA MS/MS, or at both MS1 and MS2 levels as in
MRM. By eliminating mass filtering at the MS1 level (or using large isolation windows),
multiple precursors are fragmented simultaneously and their fragments enter the second mass
analyzer simultaneously. This results in a highly complex MS/MS spectrum as the fragments
cannot initially be assigned to precursors for sequence identification. However, precursors and
fragments can be aligned by software using their retention times and mass to charge ratios (m/z).
By eliminating filtering at the MS2 level, the intensities of all of these fragments over time can be
determined for quantification. By matching precursors and fragments, then quantifying the
fragments of a given precursor, all fragmentation reactions are monitored (all reactions
monitoring, ARM). Every peptide with several transitions observed can thus be quantified.
255
Several instrument vendors have incorporated this technique into new instruments with slight
variations, and the one described and used here is Waters MSE.
This useful technique for simultaneous identification and quantification has been
conducted in a variety of samples at the protein level [2, 6-9], with each protein having multiple
peptides detected. However quantification has not previously been done with analytes at the
peptide level. In addition, the SRM software Skyline has not previously been demonstrated in
quantitation of DIA MS/MS data. In this work, the MSE strategy for DIA MS/MS is employed to
collect data for quantitation and identification of peptides, using a protein digest as internal
standard and Skyline software for data analysis. This technique is found to be linear for several
model NP standards, and proof-of-principle experiments establish its ability to quantify NPs in
tissue extracts from the blue crab Callinectes sapidus after feeding. This tool is highly useful for
function-driven
discovery
neuropeptidomics
experiments,
in
which
an
experimental
manipulation is employed to drive expression of NPs, whose identity may not already be known.
These NPs are then identified and given a putative function based on the manipulation.
6.3. Materials and Methods
More detailed information for this section is found in Appendix A.
6.3.1. Sample Preparation and Instrumental Analysis
A set of standards to test the linearity of the method was prepared using myoglobin digest
and peptide standards obtained from American Peptide Co. (Sunnyvale, CA). Equine skeletal
myoglobin (ERA, Colden, CO) was digested with trypsin (bovine pancreas, Sigma-Aldrich, St.
Louis, MO) following a published procedure [10]. Myoglobin was dissolved in 100 mM
NH4HCO3 at 0.5 mg/mL. Trypsin was dissolved in the same solution at 1 µg/mL and added to
the myoglobin solution at an enzyme: substrate ratio of 1:10. This was then diluted 1:1 with
256
methanol and placed in a water bath at 37°C for 45 min. The reaction was stopped by adding icecold acetic acid to a final concentration of 5%. The sample was then spun at 15,100 x g for 5
min. The peptide standards used were α-melanocyte stimulating hormone (MSH), bradykinin
(BK), crustacean cardioactive peptide (CCAP), Homarus americanus FMRFamide-like peptide
(FLP) I, H. americanus FLP II, somatostatin-14 (SMT), and substance P (SP). These were spiked
into a solution of 1x diluted crab saline (220 mM NaCl; 5.5 mM KCl; 6.5 mM CaCl2; 13 mM
MgCl2; 5 mM HEPES, pH 7.4) with 0.05% formic acid and 1.88 µM myoglobin digest at the
following concentrations: 100, 50, 37.5, 25, 12.5, 6.3, and 2.5 nM. These samples were not
subjected to pre-injection purification.
6.3.2. Animals and Feeding Experiments
Female blue crabs (Callinectes sapidus), were obtained from a local market. Details of
their housing and dissection are detailed in Appendix A. For animals from the fed condition, they
were presented with raw fish 1hr prior to dissection. It was confirmed that they had eaten by the
presence of food in their stomachs. Unfed animals were exposed to food but did not eat, also
evidenced by stomach contents. Four animals were used per condition. Animals were weighed
following anesthesia immediately prior to dissection. Sinus glands (SGs), brains, and pericardial
organs (POs) were isolated and stored in tubes containing 50 µL of acidified methanol (90:9:1
MeOH: H2O: acetic acid) on ice. Tissues were kept at -20°C until extraction. Following
extraction (pellet extracted 3 x 100 µL acidified methanol: grind, sonicate, spin 5 min at 16,100 x
g) and solvent evaporation (Savant SC10 Speedvac, Thermo Scientific), PO samples were using
C18 Ziptips (EMD Millipore, Billerica, MA), eluted in 15 µL of 50/50/0.1 (ACN/H2O/FA),
diluted with 5 µL.0.1% FA, and added to 2 µL of 56.4 µM myoglobin digest in LC vials. SG
257
and brain samples were resuspended in 30 µL of 0.1% FA in H2O and placed in LC vials, along
with 2 µL of 56.4 µM myoglobin digest internal standard.
6.3.3. Instrumental Analysis
Ultrahigh performance liquid chromatography (UPLC)-MSE analysis was conducted on a
Waters nanoAcquity UPLC coupled to a Waters Synapt G2 mass spectrometer (Waters, Milford,
MA). Samples (2 µL) were trapped on a preconcentration column and desalted on-line. This
column was then put in line with a C18 reversed-phase column (BEH130 C18, 1.7 µm, 75 µm x
100 mm, Waters, Milford, MA). The outlet of this column was connected to a fused silica
capillary with a pulled tip of internal diameter ~5 µm (Sutter Instrument Company, Novato, CA).
This was used as the ESI inlet. A 75 min reversed-phase run with solvent A as 0.1% FA in water
and B as 0.1% FA in ACN was used. The instrument was operated in data-independent MS/MS
mode with the high energy scan having a voltage ramp from 25 to 65 V and Glu-fibrinopeptide
was infused for lockspray calibration.
6.3.4. Data Analysis
6.3.4.1. Linearity Experiment Data Analysis
The sequences of the neuropeptide standards (CCAP: PFC(-H)NAFTGC(-H)amide, FLP
I: SDRNFLRFamide, FLP II: TNRNFLRFamide, α-MSH: acetyl-SYSMEHFRWGKPVamide,
somatostatin-14: AGC(-H)KNFFWKTFTSC(-H)amide, bradykinin: RPPGFSPFR, and substance
P: RPKPQQFGGLMamide, with (-H) indicating a C in a disulfide bond that loses a hydrogen)
and
9
myoglobin
tryptic
peptides
(FDKFK,
FKHLK,
TEAEMK,
TEAEM(Ox)K,
TEAEMKASEDLK, ALEFLR, ALEFRNDIAAK, NDIAAK, and ELGFQG, with M(O)
indicating an oxidized methionine) were input into the Skyline SRM software. Modifications
were added to peptides manually. A non-specific enzyme type was used, with cleavage at every
258
amino acid, and 9 missed cleavages permitted, the most allowed. Skyline does not have a “no
enzyme” option built in and this is the closest approximation possible. Most NPs are shorter than
9 amino acids, so this should be acceptable. In addition, Skyline will quantify peptides that do
not meet its filter criteria if they are input manually and the exception to the filters is noted by
the user. The iRT function with retention time predictor was used, calibrated off of results from
Mascot (Matrix Science, London, UK) identifications of equine myoglobin tryptic peptides in a
previous LC run of the myoglobin digest alone under the same conditions. The 2+ and 3+
precursors were monitored to the 4 most abundant 1+ and 2+ b and y ions, and in some cases
(CCAP, somatostatin-14) the precursor ion itself was also monitored. MS/MS filtering
parameters were set to DIA with MSe isolation, and 10,000 resolving power. Peak integration
was checked manually for a good match to the most abundant transitions and agreement with the
predicted retention times. Between runs, which were all conducted sequentially on the same day,
variation of less than 0.2 min was observed in retention time. Peak areas were exported to
Microsoft Excel. All transitions for each peptide were summed, and the 9 myoglobin peptides
were summed together. Peptide peak areas were normalized to the total myoglobin internal
standard peak area. Data was plotted and linear regressions were conducted.
6.3.4.2. Feeding Data Analysis
Data files were converted to .pkl files by Protein Lynx Global Server (PLGS, Waters,
Milford, MA). They were then searched against the database LiDB+crustaceanNP as defined in
Ch. 5 with Mascot (Matrix Science, London, UK). The resulting data files were used to generate
a list of observed NPs that had match scores greater than 15. A Skyline SRM method was created
that quantified all identified crab NPs from a previously published study on crab NP changes in
feeding [11]. The sequences for these were pasted in from a Microsoft Excel spreadsheet and the
259
proper post-translational modifications (PTMs) were added manually. Myoglobin tryptic
peptides quantified in the linearity experiments were also quantified in this document. Peaks
identified using Mascot were also added to the list of NPs to be quantified, with the appropriate
PTMs. The same retention time prediction was used, and again peaks were checked manually for
proper integration. However, all predicted b and y ions were monitored instead of just 4. If an ion
was observed to have a large amount of interference in its channel, it was removed. Peak areas
were exported to Microsoft Excel. Peptide peak areas were compiled and normalized to the
myoglobin peak area and the crab’s weight. From 8 crabs, the average weight ±standard error of
the mean was 190.0 ±10.3 g with the largest crab at 248.2 g and the smallest at 162.2 g. There
was no difference in weight between the fed and unfed groups, using a paired Student’s t test
(p=0.832). These normalized peak areas were compared using a paired Student’s t test and
graphed in Microsoft Excel.
6.5. Results and Discussion
6.5.1. Linearity for Quantification of Neuropeptide Standards
First, it should be noted that poor or no signal was observed for somatostatin-14 in any
sample, likely due to degradation of the standard over years. No results are presented for this NP.
All other standards were present in sufficient quantity for the peak to be integrated. Plotting the
normalized peak areas for the NP standards versus the concentrations and performing linear
regressions yielded fits with a high degree of linearity (Fig. 6.3). Values for the goodness of fit
for these regressions range from 0.93 to 0.989. These results indicate that although each analyte
is a single peptide, accurate measurement is possible by making enough measurements of its
intensity by analyzing several transitions for each of two charge states per peptide. The inclusion
of an internal standard (ITSD) with multiple peptides that can be monitored allows for even more
260
measurements of its intensity to be made, and thus this value is more reproducivle. It could also
be possible to use several peptide standards spiked in to the sample as an ITSD in a similar
manner. These results suggest that absolute quantitation of known NPs following generation of
calibration curves is also possible, although that is not carried out in this experiment. The
margins of error obtained are sufficient for relative quantitation in function-driven discovery
assays.
It should also be noted that these linearity measurements were conducted on a single set
of samples and a single technical replicate. For this to produce such highly linear results with no
outliers, the quantitation method must be very rugged. This is similar to the type of run one
might conduct in a function-driven discovery assay, where sample volume is limited and
multiple technical replicates are too consumptive of precious sample and instrument time.
Although only one experiment from a single day is shown here, an additional linearity study was
conducted using mammalian NP standards as ITSD and crustacean NP standards as analytes, and
a high degree of linearity was observed in this experiment as well. Therefore, day-to-day
reproducibility and proof of the concept that multiple peptides (whether a digest or mixture of
standards) can serve as an ITSD for reliable label-free quantitation of NPs in DIA MS/MS are
established.
6.5.2. Quantitation of Neuropeptides in Fed and Unfed Callinectes sapidus
The scheme used above with peptide standards was also used to quantify NPs from tissue
extracts of fed and unfed blue crabs to demonstrate its applicability in a function-driven NP
discovery experiment. Feeding is an important biological process known to be regulated by NPs,
and previous work in the Jonah crab, Cancer borealis, has demonstrated changes in NP levels in
the pericardial organ (PO) and brain using isotopic labeling with formaldehyde and deuterium
261
formaldehyde [11]. In the previous study, several members of the RFamide, RYamide, and
tachykinin-related peptide (TRP) families, along with the peptides proctolin (RYLPT) and
YRamide increased significantly in the brain following feeding. One peptide from the pyrokinin
family decreased in the brain following feeding. In the PO, NPs were observed to decrease
significantly, including many RFamides and one RYamide. A model in which NPs are secreted
from the PO, a known neurosecretory organ, into the hemolymph, from where they diffuse to
many target sites including the brain was one hypothetical explanation for the observed results.
Replication of these results and expansion of the list of peptides differentially regulated by
feeding was the goal of this portion of the study.
Callinectes sapidus is a commercially important crab species whose neuropeptidome has
recently been described [12, 13]. It was chosen for study due to the high degree of NP homology
and its enthusiastic feeding behavior compared to C. borealis. The NPs previously quantified
with respect to feeding mentioned above were quantified from the MSE data, along with 18 crab
NPs identified by a Mascot search with the data against a crab neuropeptide database. These
additional peptides were quantified to demonstrate the power of this technique to quantify any
NP of interest and conduct untargeted methods. In turn, this same data could be re-analyzed for
a NP found in a later study to potentially be of interest in feeding.
The peak areas were normalized to a myoglobin digest ITSD as done previously and
normalized to the weight of the crab to account for larger crabs typically having larger tissues.
The results obtained for the NPs studied previously in feeding are indicated in Fig. 6.4. Some
NPs are not detected, or are only detected at very low levels. In addition, different trends and
numbers of significant values are observed than were previously reported from feeding studies in
Cancer borealis [11]. Although the actual results are different, it is difficult to determine the
262
relevance of this finding, as different species of crabs and methods of MS were used. What can
be determined is that most of the NPs were detected and quantified, and values obtained do not
have a high degree of error. Statistically significant p<0.05) increases were seen in the SG for an
orcokinin NFDEIDRSGFamide and a pyrokinin SGGFAFSPRLamide. Results obtained for
quantification of the peptides identified by Mascot are shown in Fig. 6.5. Several of these NPs
had statistically significant changes, and many were quantified with low error. Several of the
NPs are modified forms of each other, and those that have similar sequences also show similar
trends. Significant changes were observed for 11 NPs in the PO. This is likely due to better
identification of a putative NP from this tissue because of its rich NP content. Proof-of-principle
experiments demonstrate that this method can be used to quantify NPs from the crab resulting
from experimental manipulation in an untargeted manner.
6.6. Conclusions and Future Work
In this chapter, a method for quantitation of DIA MS/MS data in a pseudo-MRM manner
was developed. It used an open-source, vendor-neutral software for accurate identification of
precursors and fragments and integration of the resulting peaks. This quantitation method shows
linearity for mixtures of peptide standards over the range 2.5-100 nM using a myoglobin digest
as an internal standard. A proof-of-principle experiment was also conducted to quantify 28 NPs
previously associated with feeding and 18 crab peptide sequences identified from the data by a
database searching method. It is thus possible to take samples from any species for which a
database of the proteins or peptides of interest exists and quantify compounds at the peptide level
using this method. If a database does not exist, a homologous species can be used. This method
will be of great use in targeted and untargeted functional analysis studies of NPs.
6.7. Works Cited
263
1. Bantscheff, M.; Lemeer, S.; Savitski, M. M.; Kuster, B. Quantitative mass spectrometry in proteomics: critical
review update from 2007 to the present. Anal. Bioanal. Chem. 2012, 404, 939-965.
2. Gillet, L. C.; Navarro, P.; Tate, S.; Rost, H.; Selevsek, N.; Reiter, L.; Bonner, R.; Aebersold, R. Targeted Data
Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for
Consistent and Accurate Proteome Analysis. Molecular & Cellular Proteomics. 2012, 11.
3. MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.;
Liebler, D. C.; MacCoss, M. J. Skyline: an open source document editor for creating and analyzing targeted
proteomics experiments. Bioinformatics. 2010, 26, 966-968.
4. Xiang, F.; Ye, H.; Chen, R.; Fu, Q.; Li, L. N,N-dimethyl leucines as novel isobaric tandem mass tags for
quantitative proteomics and peptidomics. Anal Chem. 2010, 82, 2817-2825.
5. Sherrod, S. D.; Myers, M. V.; Li, M.; Myers, J. S.; Carpenter, K. L.; MacLean, B.; MacCoss, M. J.; Liebler, D.
C.; Ham, A.-J. L. Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction
Monitoring with Internal Reference Peptides. Journal of Proteome Research. 2012, 11, 3467-3479.
6. Geromanos, S. J.; Vissers, J. P. C.; Silva, J. C.; Dorschel, C. A.; Li, G.-Z.; Gorenstein, M. V.; Bateman, R. H.;
Langridge, J. I. The detection, correlation, and comparison of peptide precursor and product ions from data
independent LC-MS with data dependant LC-MS/MS. Proteomics. 2009, 9, 1683-1695.
7. Levin, Y.; Hradetzky, E.; Bahn, S. Quantification of proteins using data-independent analysis (MSE) in simple
andcomplex samples: a systematic evaluation. Proteomics. 2011, 11, 3273-3287.
8. Martins-de-Souza, D.; Guest, P. C.; Guest, F. L.; Bauder, C.; Rahmoune, H.; Pietsch, S.; Roeber, S.; Kretzschmar,
H.; Mann, D.; Baborie, A.; Bahn, S. Characterization of the human primary visual cortex and cerebellum
proteomes using shotgun mass spectrometry-data-independent analyses. Proteomics. 2012, 12, 500-504.
9. Mbeunkui, F.; Goshe, M. B. Investigation of solubilization and digestion methods for microsomal membrane
proteome analysis using data-independent LC-MSE. Proteomics. 2011, 11, 898-911.
10. Li, F.; Schmerberg, C. M.; Ji, Q. C. Accelerated tryptic digestion of proteins in plasma for absolute quantitation
using a protein internal standard by liquid chromatography/tandem mass spectrometry. Rapid Commun
Mass Spectrom. 2009, 23, 729-732.
11. Chen, R.; Hui, L.; Cape, S. S.; Wang, J.; Li, L. Comparative Neuropeptidomic Analysis of Food Intake via a
Multi-faceted Mass Spectrometric Approach. ACS Chem Neurosci. 2010, 1, 204-214.
12. Hui, L.; Cunningham, R.; Zhang, Z.; Cao, W.; Jia, C.; Li, L. Discovery and characterization of the Crustacean
hyperglycemic hormone precursor related peptides (CPRP) and orcokinin neuropeptides in the sinus glands
of the blue crab Callinectes sapidus using multiple tandem mass spectrometry techniques. J Proteome Res.
2011, 10, 4219-4229.
13. Hui, L.; Xiang, F.; Zhang, Y.; Li, L. Mass spectrometric elucidation of the neuropeptidome of a crustacean
neuroendocrine organ. Peptides. 2012, 36, 230-239.
264
6.7. Figures
Figure 6.1. Multiple reaction monitoring (MRM) MS/MS. In the first MS, the precursors are
detected and a single one is selected by mass filters to enter the collision cell. There, it collides
with gas and undergoes collision-induced dissociation (CID). A single fragment ion of interest is
then sent to the second MS analyzer, where it is detected. Setting the mass filters to the
appropriate m/z ratios leads to highly efficient and reproducible transfer of ions from MS 1 all the
way to the detector. In the duty cycle of the instrument, the masses chosen in MS1 and MS2 can
be rapidly changed (on the order of msec) to allow a different precursor and fragment to be
analyzed.
265
Figure 6.2. Data-Independent Analysis (DIA) MS/MS. DIA MS/MS, called MSE on Waters
QTOF instruments, has two spectral acquisition modes alternating throughout the entire
acquisition. A low energy spectrum is generated by allowing just precursors to be analyzed. In a
high energy spectrum, all precursors are subjected to CID simultaneously, and then all fragments
are analyzed. Precursors and fragments are then aligned to each other based on a number of
parameters, most notably their LC retention time.
266
Figure 6.3. Linearity of normalized peptide standard responses. The linearity of quantitation
using multiple myoglobin tryptic peptides as internal standards was determined for 6 NP
standards (abbreviations in text). Good linearity was observed in the 2.5-100 nM range (5 to 200
fmol on column) with ITSD at 1.88 µM.
0.04
Area/Area ITSD
Area/Area ITSD
RPPGFSPFRamide
PFC(-H)NAFTGC(-H)amide
0.05
0.03
0.02
y = 0.0004x + 0.0081
R² = 0.9558
0.01
0
0
50
100
Concentration (nM)
150
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
y = 0.0006x + 0.0038
R² = 0.9625
0
150
Ac-SYSMEHFRWGKPVamide
SDRNFLRFamide
Area/Area ITSD
Area/Area ITSD
0.05
0.03
0.025
0.02
0.015
0.01
0.005
0
0.04
0.03
0.02
y = 0.0002x + 0.0033
R² = 0.9644
0
50
100
Concentration (nM)
y = 0.0004x + 0.0014
R² = 0.989
0.01
0
150
0
50
100
Concentration (nM)
RPKPQQFFGLMamide
150
TNRNFLRFamide
0.1
Area/Area ITSD
0.05
Area/Area ITSD
50
100
Concentration (nM)
0.04
0.03
0.02
y = 0.0003x + 0.0145
R² = 0.93
0.01
0.08
0.06
0.04
y = 0.0008x + 0.0024
R² = 0.977
0.02
0
0
0
0
50
100
Concentration (nM)
150
50
100
Concentration (nM)
150
267
Figure 6.4. Changes in NP levels following feeding in three tissues. NPs quantified here were
chosen based on their inclusion in a previous feeding study in the crab [11]. Means and standard
errors of the means are indicated. Statistically significant changes between the two conditions are
indicated
with
an
asterisk
(*p<0.05).
These
are
on
NFDEIDRSGFamide
SGGFAFSPRFamide for the sinus gland. Four animals were used in each condition.
and
268
0.0012
A) Pericardial Organ
Fed
Unfed
0.001
0.0008
0.0006
0.0004
0.0002
0
0.0012
B) Brain
0.001
Fed
Unfed
0.0008
0.0006
0.0004
0.0002
0
0.006
C) Sinus Gland
0.005
Fed
0.004
Unfed
*
0.003
*
0.002
0.001
0
-0.001
269
Figure 6.5. Changes in NP levels following feeding in three tissues. NPs quantified here were
chosen based on an untargeted approach. Data was searched against a crab NP database and the
resulting NPs with scores>15 that were not very large were quantified. Means and standard
errors of the means are indicated. Statistically significant values are indicated with an asterisk (*
p<0.05). Four animals were used for each condition.
270
0.007
A) Pericardial Organ
*
0.006
*
0.005
Fed
0.004
Unfed
0.003
0.002
0.001
*
*
*
*
*
*
*
*
0
0.02
0.018
0.016
0.014
0.012
0.01
0.008
0.006
0.004
0.002
0
*
B) Brain
Fed
Unfed
271
0.05
0.045
C) Sinus Gland
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
Fed
Unfed
272
Chapter 7: Application of Microdialysis and Data-Independent MS/MS Quantitation for
Determination of Feeding-Related Neuropeptides
Adapted from Schmerberg, C. M. and Li, L. ACS Chem. Neurosci. [in preparation]
7.1. Abstract
Food consumption is an important behavior for sustaining the life of an animal that is
regulated by an intricate array of neuropeptides (NPs) in many species. Although much is known
about the identities of these NPs in mammals, the exact mechanisms of how these multiple
chemical entities act on several feeding-related neuronal circuits to generate feeding behavior is
currently unknown and difficult to study systematically in mammals. In order to better
understand dynamic changes in NPs during feeding behavior, a decapod crustacean model
system was employed in conjunction with novel microdialysis (MD) and data-independent
acquisition (DIA) tandem mass spectrometry (MS/MS) quantitation and identification methods.
The simpler neurochemistry and neural circuitry of the decapod crustacean make it a potentially
useful model for elucidating the effects of some of these NPs, as well as a successful model
animal for analytical chemistry method development. MD samples were collected from the
Jonah crab, Cancer borealis, throughout the time course of feeding and analyzed for NP content
in an untargeted quantitative MS/MS method that also provides peptide sequence information. A
total of 28 NPs previously identified as potentially related to feeding in previous studies were
quantified, and 5 of those showed statistically significant fold-changes: APQRNFLRFamide,
GAHKNYLRFamide, GPRNFLRFamide, SENRNFLRFamide, and HIGSLYRamide. Some
general trends were observed, particularly an immediate release into the hemolymph in the first
30 min after feeding, followed by an overshoot and return to baseline. A variety of NP level
changes were observed in the time period much later after feeding, indicating that the initial roles
273
for these NPs may be similar, but the long-term roles may be different. This work demonstrates
the success of this technique for quantifying NPs in a behavioral neuroscience experiment and
establishes putative roles for several NPs in feeding behavior.
7.2 Introduction
Neuropeptides (NPs) are known to play an important role in regulation of feeding
behavior in a variety of species. Several NPs have been identified as modulating food
consumption on a number of different levels in mammalian systems, including galanin, ghrelin,
neuropeptide Y, melanin-concentrating hormone (MCH), alpha-melanocyte stimulating hormone
(α-MSH), orexins, oxytocin, insulin, leptin, cholecystokinin (CCK), agouti-related protein
(AgRP), neurotensin (NT), neuropeptide W, neuropeptide YY (PYY) and cocaine and
amphetamine regulated transcript (CART) [1-3]. These NPs can act locally as neurotransmitters
or at a distance in an endocrine manner [4, 5]. Some NPs are implicated in feeding-related
modulation of the brain’s endogenous reward pathways, including circulating hormones and
locally acting neuropeptides [1]. Several of the circulating hormones’ levels are controlled
primarily by other organs, such as insulin from the pancreas, leptin from fat cells, and ghrelin
from the stomach, and thus they and other NPs can serve as a way for the body to monitor energy
levels and stores and alter feeding behavior accordingly [6, 7]. Many NPs have been identified in
mammals as important in feeding behavior through a number of pathways.
Although much progress has been made in understanding the effects of NPs on
consumption behavior, the mechanisms by which a chemical signal acts to produce a given
behavior are not well understood. One challenge lies in understanding behavior generation in the
complex brains of traditional animal models; another lies in difficulties to accurately characterize
NP changes on a physiologically relevant time scale. A number of diverse signals converge on
274
feeding behavior, including reward circuitry, stress signaling, metabolic state signals,
somatosensation, and multiple hypothalamic subnuclei [2]. Put simply, the decision to eat is
influenced not only by how “hungry” one is (stomach fullness and/or circulating glucose levels),
but also how much one derives pleasure from food, environmental effects (stress, circadian), the
type of food, whether other rewarding stimuli are present, etc. A major concern in this area of
research is dissecting the pathways of feeding for energy maintenance and feeding for pleasure
(homeostatic vs. hedonic), and it appears NPs are involved in both pathways in mammals [3].
Secondly, NPs can be difficult to quantify accurately due to the presence of alternative splice
forms and antibody cross-reactivity, and accessing them on a physiologically relevant time scale
can also be a challenge [8].
With a simple nervous system and more limited suite of behaviors, decapod crustaceans
are potentially an attractive model for ingestive behavior. Food is highly motivating for
crustaceans, and they can even be conditioned to press a lever to receive a food reward [9]. The
crustacean stomatogastric ganglion (STG) and other ganglia are part of well-defined neural
circuits responsible mainly for rhythmic movements of the gut in the crab. This well-established
model for understanding rhythmic neuron firing and the effects of neuromodulators on activity
has been extensively studied and can be modeled using computational neuroscience [10, 11].
Thus, putative NPs can be applied in this system experimentally, and models can be used to fit
the experimental evidence. This, in turn, can provide information about the components of the
circuit by which the NP acts, for instance, changing the current flux through one particular type
of ion channel. Certain firing patterns in this circuit are associated with chewing or filtering of
food, and thus can be correlated directly to feeding behavior. In addition to possessing this set of
useful tools for understanding NP action on the circuit and ion channel level, the decapod
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crustacean contains several organs that are rich in NPs that can be analyzed using well-developed
mass spectrometry (MS)-based techniques [8].
Mass spectrometry-based techniques for NP analysis are more specific than antibodybased methods due to the ability to characterize an analyte based on its mass to charge ratio
(m/z) and its fragments’ m/z. MS-based techniques can also be highly accurate and multiplexed,
as evidenced in Chapter 6. Microdialysis (MD) sampling of NPs allows for collection of NPs
concurrent with performance of normal behaviors, so that a close correlation can be made
between changes in NP levels and behavioral changes. These techniques have been developed
extensively in the decapod crustacean due to the reduced complexity of its neurochemical
environment and neuronal circuits [12, 13]. In this chapter, techniques developed in previous
chapters were applied to study NP changes in crabs during feeding behavior. Microdialysis was
used to collect NPs from the hemolymph of Jonah crabs, Cancer borealis, before, during, and up
to 3 hrs after feeding. These samples were then subjected to data-independent analysis (DIA)
tandem MS (MS/MS) to enable determination and/or verification of NP sequences and highly
specific quantity changes in the same instrument run. The resulting data was analyzed to quantify
28 NPs previously studied with regards to feeding in C. borealis [14]. The method was
successful in identifying statistically significant changes in abundance at 30-min interval time
points
for 5
of the NPs
quantified.
These included the RFamide-like peptides
APQRNFLRFamide, GAHKNYLRFamide, GPRNFLRFamide, and SENRNFLRFamide; and
the YRamide HIGSLYRamide. Overall profiles for the NPs in hemolymph appear to have initial
release events, followed by a return to baseline levels, although there are notable differences in
some NPs. These changes suggest putative roles for these NPs in the neuroendocrine control of
feeding behavior and physiological changes associated with ingestion. These findings are
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consistent with previous tissue extract studies of feeding-related NPs in crabs. This is the first
quantitative assessment of NPs in dialysates from the crab and demonstrates the utility of MD
and MS/MS-based tools for behavioral neuroscience studies of NP function.
7.3. Methods
Several of the methods have been described previously in Chapters 4 and 6, and are
presented in detail in Appendix A. Only where the methods differ from previous chapters will
they be discussed here.
7.3.1. In Vivo Microdialysis
PAES (20kDa MWCO) probes (CMA Microdialysis) were implanted in Cancer borealis
as indicated in Appendix A and Chapter 4. Following implantation, crabs with probes were kept
separate from other animals with the aid of plexiglass dividers that the water could flow through
and around. The outlet of the probe was connected to an automated refrigerated fraction
collector. Animals were allowed to recover a minimum of 24 hrs prior to collecting samples that
were used for analysis. Crabs typically would not eat until a minimum of 48 hrs had elapsed after
surgery, so feeding experiments were conducted beginning the 2nd day after surgery. Crab saline
was infused through the system at a flow rate of 0.5 µL/min, and the probe was allowed to run at
this flow rate for a minimum of 30 min before any samples were collected to permit
equilibration. In many cases, the experiment was started after the perfusate had been running at
that rate for several hours and equilibration was already complete. If the rate had to be changed
or the syringe refilled, 30 min of equilibration time immediately preceded collection of samples
for the feeding experiment. The dead volume of the probe system from the tip of the probe to the
collection tube was calculated based on information from the manufacturers of the probe, tubing,
and fraction collector, and this was taken into account for timing collections correctly with
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feeding. Crabs were fed white-fleshed raw fish (cod, tilapia, etc.), sold for human consumption,
thawed and cut into small pieces immediately prior to use. Most crabs took and started eating the
food immediately. If it took more than 2-3 min for the crab to start eating, the experiment was
abandoned for that day. For crabs that did eat, excess food was always present so that the animal
could feed until satiety was reached. Crabs typically stopped eating after 45-60 min. At t = 180
min, any remaining food was removed from the tank, even if the crab had to be moved to retrieve
it. A second feeding experiment was conducted with each crab (three crabs total) a minimum of
24 hrs after the start of the first one. Crabs were not otherwise disturbed at any point during this
process, and the room was consistently kept under red light to simulate night conditions.
For every experiment, two baseline samples were taken from t = -60min to t = -30 min
and t = -30min to t = 0 min. Collection tubes were changed every 30 min during the experiment
by the refrigerated fraction collector until 8 samples were obtained, for a final time of 180 min
post-feeding. Samples are graphed according to the time midway through their collection time.
The resulting 15 µL samples were immediately acidified by adding 1.5 µL pure formic acid (FA)
to them, bringing their acid content to approximately 10%, as this has been shown to reduce NP
degradation [15]. They were then kept at -20°C until they could be analyzed. The feeding
experiment was conducted twice for each crab with three crabs for a total of 6 replicates (3
biological x 2 experimental).
7.3.2. Instrumental Analysis
Samples were placed in LC vials and an internal standard (1 µL of 56.4 µM myoglobin
digest solution prepared as in Protocol A and Chapter 6) was added. Immediately prior to
analysis on UPLC-MS/MS, 1 µL of a 0.91 M NH4HCO3 solution was added to increase the
sample’s pH so as not to damage the UPLC column. Samples were analyzed as in Chapter 6 and
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Protocol A, with a Waters nanoAcquity UPLC coupled via electrospray ionization (ESI) to a
Waters Synapt G2 QTOF operating in MSE mode. Reversed-phase LC runs were 75 min long for
each run.
7.3.3. Data Analysis
A previously developed method for quantification of MSE data (Chapter 6) using Skyline
software (University of Washington [16]) was used. A list of 28 NPs previously observed in
feeding experiments (enumerated in Table 7.1.) [14] along with 9 myoglobin tryptic peptides
(FDKFK, FKHLK, TEAEMK, TEAEM(O)K, TEAEMKASEDLK, ALEFLR, ALEFRNDIAAK,
NDIAAK, and ELGFQG, with M(O) indicating an oxidized methionine) was selected for data
extraction and integration. The iRT function with retention time predictor was used, calibrated
off of results from Mascot (Matrix Science, London, UK) identifications of equine myoglobin
tryptic peptides in a previous LC run of the myoglobin digest alone under the same UPLC
conditions. The 2+ and 3+ precursors were monitored for their transitions to the most abundant 1+
and 2+ b and y ions. MS/MS filtering parameters were set to DIA with MSe isolation, and 10,000
resolving power. Peak integration was checked manually for a good match to the most abundant
transitions and agreement with the predicted retention times. Variation was greater between sets
of samples run on the instrument on different days than within a single day. All samples from a
given MD experiment were run on the instrument on the same day, and analyzed in parallel.
Peak areas were exported to Microsoft Excel. All transitions for each peptide were summed, and
the 9 myoglobin peptides were summed together. Peptide peak areas were normalized to the total
myoglobin internal standard peak area. The two baseline values were averaged for each
experiment, and all values were normalized to this to correct for variability between animals and
between different days. Thus, data represents fold-changes from baseline.
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Results were plotted using Microsoft Excel and JMP statistical software (Version 9.0.2
SAS Institute, Inc., Cary, NC, USA). A few outlier data points were observed, likely due to
signal interference in UPLC-MS/MS or improper peak picking, so data points of >5-fold or <0.2fold compared to baseline were removed. The most data points removed from any given peptide
for any time point was 2, and no two experimental replicates were removed for a single crab, so
there were always 3 biological replicates at every data point. The validity of repeated measures
was determined using multivariate analysis in JMP. Briefly, a multiple analysis of variance
(MANOVA) and multivariate repeated-measures analysis were conducted, and the validity of the
measurements made was assessed based on these tests. A one way analysis of variance
(ANOVA) comparison was conducted, followed by a Tukey-Kramer Honestly Significant
Differences (HSD) all pairs test at an alpha value of 0.05. p-Values that were less than 0.05 were
considered significant. Means and standard errors of the means (SEM) for all NPs were graphed
in Microsoft Excel, with p-values from the Tukey-Kramer HSD test superimposed on the graph
for significant values as indicated.
7.4. Results and Discussion
The method was successful at generating reproducible measurements of NPs in multiple
samples from 2 experimental replicates with each of 3 different animals. The distribution of
outliers is illustrated by Fig. 7.1.A, which a scatterplot of all data points before outliers
representing a 5-fold change were removed. Neuropeptide values that had such a high foldchange typically represented chromatograms where either the incorrect peak was integrated, or
the peak was not observed at all, and as such are due to integration error. A total of 22 outlying
points out of 1344 total, or 1.34%, were removed by this criterion, and the resulting scatterplot is
shown in Fig. 7.1.B. The outliers were likely a result of poor peak detection due to interference
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or integration of the incorrect peak. Removal of outliers did not reduce the total number of
biological replicates, and although variability is still observed after removal of the egregious
outliers, it is greatly reduced.
To determine the validity of making multiple repeated measures in each experimental
replicate, a multivariate repeated-measures analysis was conducted in JMP on a multiple analysis
of variance test (MANOVA). Between experiments, no significant difference was found in NP
profiles (prob>F 0.5688). Within experiments, sphericity was met, so multivariate F tests and
adjusted univariate F tests were appropriate. These determined that overall, all interactions
within subjects and of time*peptide were non-significant (F-values>0.75 for adjusted univariate
F tests). However, the time factor did cause significant differences (p<0.0001 for the multivariate
F test and univariate Greenhouse-Geisser- and Huynh-Feldt-adjusted F tests). These tests verify
that there was no bias introduced by quantifying multiple peptides over time. The NP profiles
were not significantly different between experiments, and overall interactions between NPs were
non-significant. The observed variation between NP profiles was not dependent on experimental
replicate and was instead related to time. Therefore, it is valid to quantify changes in these NPs
over time and suggest that observed changes are a result of changing internal conditions in the
animals over time.
Of the 28 NPs quantified from 9 different families, significantly different values were
obtained for time points in 5 of them, from the RFamide and YRamide families. Although all 14
of the 28 were observed to change in tissue extract studies, a more limited number of biological
replicates were conducted for this experiment due to the difficulty of experimental procedures,
expense of materials (MD probes), and availability of MS/MS instrument time (each experiment
takes 12 hours of instrument time to run). Therefore, more variability is observed and fewer NPs
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have large, significant changes. The repetition of this experiment should provide additional
information about each data point, perhaps yielding more significantly changed values. For this
reason, overall trends in NP concentration profile will also be discussed, even for values where
statistical significance at the p<0.05 level has not been reached. No baseline values were
statistically significant from each other. Detailed results and graphs for each peptide are included
in this report. The peptides will be grouped and discussed by family.
7.4.1. RFamide-Like Peptides
Peptides of the RFamide-like peptide family were altered the most. Of 10 peptides
analyzed from this family, 4 had statistically significant changes. RFamide peptides are one of
the largest groups of NPs in crustaceans, with 41 individual peptides of this family described in
Cancer borealis (Chapter 2, [17, 18]). There are several known subgroups of RFamides,
including the neuropeptide F (NPF)/short neuropeptide F peptides (sNPF), which carry a Cterminal motif of RXRFamide; the myosuppressins, which have HVFLRFamide at the Cterminus, and the sulfakinins, which have a sulfated tyrosine and the C-terminal sequence
HMRFamide. Additional C-terminal sequences observed include FLRFamide, YLRFamide, and
FVRFamide [19]. For the RFamide-like peptides, the hemolymph concentrations following
feeding have a profile that can be largely described as release, an overshoot, and then eventual
return to baseline.
One peptide, GAHKNYLRFamide, had several significantly different values,
summarized in Table 7.3. This peptide was first identified in 2006 from the POs and
stomatogastric nervous systems of several Cancer species (C. borealis, C. productus, and C.
magister) [20]. The value obtained in the first 30 min after feeding was greater than the values
obtained 90-120 min, 120-150 min, and 150-180 min after feeding. The concentration at 60-90
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min post-feeding was also different from the value 150-180 min after feeding. This is illustrated
in Fig. 7.2. There are two clear phases in the hemolymph concentration of this peptide. Shortly
after feeding, levels become higher than baseline by 20-25%. Much later, they drop back down
to baseline or 10-15% less than baseline. This suggests that GAHKNYLRFamide is released
during active feeding behavior and is returned to baseline levels shortly after feeding has
stopped. Although the only statistical difference is between the time points indicated, the overall
profile potentially shows an initial release that is stopped after 30 min (the lower value for
45min). A second release event may occur around 75 min, and after another 30 min period, the
levels are low again, perhaps even lower than they were before feeding. The potentially reduced
level at 45 min and the clearly reduced level starting at 105 min may indicate a depletion of PO
stores of this peptide, supported by previous studies finding a statistically significant 43%
decrease in its levels in this tissue 30-45 min after feeding [14]. In the same study, this peptide
was also observed to increase in brain tissue during this time period, so it may be being bound
and internalized with receptors as part of a desensitization mechanism in this tissue. This nonsignificant profile matches with a two-stage feeding behavior that is typically observed in this
and other crab species, although it has not been studied systematically. Crabs typically start
eating immediately and stop after 30 min, then wait another 15-25 min and start eating again in a
shorter feeding period. This could mirror the two observed increases in GAHKNYLRFamide as
they occur around the times that each of these feeding periods begins. In any case, the
hemolymph levels remain elevated for a while after feeding begins, so its degradation must be
slower than its release for a long period of time. This also suggests that GAHKNYLRFamide is
not initiating feeding, but may be sustaining the behavior or serving as a whole-body signal that
food is being taken in, as a mechanism of coordinating multiple organ systems.
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Another RFamide with statistically significant changes in its levels after feeding is
SENRNFLRFamide (Fig. 7.3, Table 7.4). This peptide has been found in many crustacean
species, but was first identified in 2005 from the PO of the crab Cancer productus [21].
SENRNFLRFamide is more concentrated in hemolymph during the time 0-30 min after feeding
compared to 90-120 min and 120-150 min after feeding. Therefore, a reduction in this peptide
appears after the animal has stopped eating. The non-significant trend observed is a rapid
increase immediately after feeding that drops back to baseline levels or lower after more than 90
min following feeding. This peptide could have similar functions as described for
GAHKNYLRFamide. Reduced levels in the hemolymph at later times could also be due to
depletion of stores, similar to observations in PO tissue extract studies [14]. This peptide did not
have a significant increase in brain concentration after feeding in those studies, so the brain may
not be its primary site of action, or it may be degraded by extracellular peptidases instead of
internalized while bound to its receptor as part of desensitization.
A third RFamide, GPRNFLRFamide, was significantly higher in concentration
immediately after feeding when compared to more than 2 hrs after feeding (Fig. 7.4, Table 7.5).
This peptide was initially identified in Cancer borealis after immunoaffinity enrichment of
extracts from its POs [17]. The difference in values was around 80% of the baseline value, so
this change is large in magnitude. In general, it appears that this peptide is released immediately
after feeding and then may rapidly return to baseline or sub-baseline levels. Thus, it may be an
indicator of the beginning of the feeding process that could trigger release of other compounds
with more gradual action, or it could be active in initiating feeding behaviors. This peptide was
detected in tissue studies, and showed a strong but not statistically significant trend toward a
small decrease in PO extract, but was not significantly changed in either tissue [14]. Thus, it may
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be released by other organs and act outside of the brain. Its degradation or sequestration from the
hemolymph appears to be quite rapid.
The final RFamide-like peptide with significant changes in concentration is
APQRNFLRFamide, also known as Procambarus clarkii RFamide-Related Peptide 6 (PrcFaRP
6). It was identified first in Cancer borealis in 2003 [22], but received this name in 2006 after it
was found in P. clarkii in 2006 [23]. The results for this peptide are illustrated in Table 7.6 and
Fig. 7.5. This peptide’s concentration is significantly changed between the samples taken 0 to 30
min after feeding and 90 min to 120 min after feeding, a profile very similar to
GPRNFLRFamide. The overall concentration profile of APQRNFLRFamide is not similar,
however, as it appears that this peptide is released immediately after feeding, decreases below
baseline after 2 hrs, and then returns to a baseline value. As with SENRNFLRFamide and
GPRNFLRFamide, this profile indicates that the peptide may be important in initiating feeding
behavior but not necessarily for sustaining it, unlike GAHKNYLRFamide. It is interesting to
note that 3 of the peptides with significant changes have the extended N-terminal motif of –
RNFLRFamide, which has not previously been suggested as a functionally relevant feature.
However, additional peptides with this sequence at the C-terminus were quantified in this study
and did not show significant changes, so this may be coincidental.
For the remaining 5 FMRFamide-related peptides quantified in this study, overall trends
may be discussed, but no statistically significant values were identified. These are illustrated in
Fig. 7.6. The same general trend is observed with these peptides—an immediate release followed
by an overshoot period and a recovery to baseline values. Although most peptides of this family
have the same general concentration profile, there are slight individual variations in timing of the
start of the initial spike, in how long the initial spike in concentration lasts, how low the
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concentration reaches in the post-spike period, and how much the baseline value is recovered
after the 180 min experiment. Slight differences in these may be important for understanding the
functions of RFamides in feeding. For instance, GYSKNYLRFamide and NRNFLRFamide
appear to have a slightly more sustained release when compared to AYNRSFLRFamide and
GNRFLRFamide. Rapid decreases in hemolymph concentration could be due to active
degradation or endocytosis, and other peptides might be present longer to either act in a similar
way to prolong the signal, or to act in a different way for a slight change in behavior, i.e. feeding
initiation vs. sustaining. What is clear, however, is that the RFamide family peptides change
dramatically after feeding, including 4 that were observed to have statistically significant
changes in this study. These peptides have been shown to alter neuronal and muscle firing [19],
and may make contractions of stomach muscles more easily initiated by motor neuron signals.
The function of many RFamides is still unknown, and additional putative roles include rewardlike function for those similar to neuropeptide F, which is analogous to neuropeptide Y in
mammals and has been shown to be important in relaying a natural reward signal in insects [24].
7.5.2. HIGSLYRamide
The peptide HIGSLYRamide was first identified in Cancer productus in 2005 [21], but
has since been detected in other crab species. No putative function for this peptide exists, and no
other YRamide peptides have been identified. This peptide was significantly increased in
hemolymph in the time period 0-30 min after feeding compared to both the first time period (-60
to -30 min relative to feeding) and a later time period (120 to 150 min after feeding) (Fig. 7.7 and
Table 7.7). This indicates that this one time period, immediately after feeding, is a time in which
this peptide changes dramatically. The increase observed is approximately 100%. Although the
graph shows that this peptide likely returns to baseline levels quickly, a statistically different
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value for its concentration is not achieved until the 120 to 150 min time point. This may indicate
some variability between animals in the rate at which this peptide’s concentration decreases
following the initial spike. This finding is consistent with a large increase in the brain seen in the
previous feeding study analyzing tissue extracts [14]. It thus appears that a large amount of this
peptide goes into circulation immediately following feeding, and it may end up in the brain via
receptor binding. Since the level is unchanged in PO, it is possible that another source releases
this peptide. The rapid return to baseline levels may indicate that this peptide is involved in the
initial stages of feeding and is less important later. This dramatic and statistically significant
change in HIGSLYRamide may indicate that this peptide is important immediately after feeding
in the crab.
7.5.3. Orcokinins
Orcokinins are a large group of hindgut-stimulating peptides [19]. In this study, none of
the 7 quantified were observed to change significantly after feeding (Fig, 7.8). In general, their
levels were steady over time, with a light possible increase in the first 30 min sample. In
previous studies, they have been found to increase slightly or be unchanged in the brain [14]. The
ability to detect and quantify these peptides in microdialysates is a great advancement, however,
over previous techniques. Analysis of hemolymph led to the detection of just one orcokinin [25],
and previous analysis of microdialysates collected over long time periods (4-18 hrs) and heavily
concentrated prior to analysis led to the detection of 6 orcokinins, 3 of which were also analyzed
here. The observation of 4 different orcokinins and the ability to quantify them reproducibly in
samples collected over only 30 min demonstrates the power of the methodology used in this
study. These peptides do not appear to be involved in feeding behavior, however, from the
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results of this and previous studies. This is surprising due to their known activity on the hindgut
of some crustaceans.
7.5.4. Tachykinin-Related Peptides (TRPs)
The tachykinin-related peptides are well-known and highly conserved NPs [19]. These
peptides have structural homology to mammalian tachykinins, from whence they derive their
name. Three are known to exist in Cancer borealis, and two are quantified here. Cancer borealis
tachykinin-related
peptide
(CabTRP)
Ia
(APSGFLGMRamide)
and
CabTRP
II
(TPSGFLGMRamide) do not change significantly throughout the course of feeding (Fig. 7.9).
However, an interesting trend is observed that may be significant upon further study.
APSGFLGMRamide appears to have its peak value in the 0 to 30 min time point, whereas the
peak for TPSGFLGMRamide occurs in the 30 to 60 min time point. It also appears to return to
baseline more gradually. This may indicate a sequential release of these related peptides, with
roles in feeding that must be carried out in a particular order. Both peptides increase dramatically
in the brain after feeding [14]. They have been shown to be released at the synapse, from
neuroendocrine organs, and from midgut epithelial cells, so a feeding-related increase in the
hemolymph and brain may suggest that these sites are releasing large amounts of stored TRPs
into the circulation. From there, they can act on the brain and perhaps on other target sites.
Evidence in insects discussed in Chapter 2 suggests a pro-feeding role for tachykinins in the
hemolymph, including stimulation of feeding in the cockroach [26]. The different profiles of
TRP levels in hemolymph for the two peptides quantified, added to previous evidence of
feeding-related increases in the brain and endocrine release of these peptides, suggest that TRPs
may be important communication molecules between the gut and/or neurosecretory organs and
the brain.
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7.5.5. RYamides
The RYamides quantified in this study, FVGGSRYamide and SGFYANRYamide, did
not significantly change throughout the course of feeding (Fig. 7.10). However, they do have a
trend similar to other peptides in this study: a rapid increase immediately after feeding, followed
by a return to baseline. The reduction in these peptides later after feeding appears to be more
gradual than for other peptides, and they have a larger fold-change. In previous studies, their
decrease in the PO was not significant, but an increase in the brain was [14]. As the function of
RYamides in crustaceans is as yet unknown, the significance of these findings is unclear.
However, there is a non-significant suggestion from this work that these peptides are released
into the hemolymph following feeding in large amounts and are eliminated from circulation
more gradually than other peptides. One of the sites where they might act after this release is the
brain.
7.5.6. B-Type Allatostatins
Two B-type allatostatins, STNWSSLRSAWamide and VPNDWAHFRGSWamide, were
characterized in this work. They were not observed to change significantly throughout the course
of feeding (Fig. 7.11). Although they have been observed in tissue extract studies of feeding,
they were not observed to change significantly [14]. As little is known about the function of
these peptides, these results provide information only that they are not likely to be involved in
feeding regulation.
7.5.7. Other Crustacean Neuropeptides
Two other peptides that are commonly found in many crustaceans were quantified (Fig.
7.12): crustacean cardioactive peptide (CCAP, PFC(-H)NAFTGC(-H)amide, (-H) indicates a
Cys in a disulfide bond) and proctolin (RYLPT). Neither had statistically significant changes in
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concentration after feeding. CCAP followed a concentration profile similar to that observed for
GAHNYLRFamide, and proctolin was unique among the peptides quantified in that it did not
return to baseline levels in later time points. Proctolin’s action in defined neural circuits has been
studied in detail (See Chapter 2, [19]), and in general it increases the activity of excitable cells.
CCAP has been implicated in feeding, particularly in insects (See Chapter 2), and has an
interesting bi-modal effect on crustacean neurons and muscles. At low hemolymph
concentrations, it affects muscle activity, and at higher concentrations, it alters neuronal activity.
Both peptides were observed to increase in the brain in a previous feeding study, but the increase
was not significant [14]. Proctolin’s concentration profile suggests long-lasting changes related
to feeding, and as such it may be acting more as an indicator of the animal’s satiety or available
energy store level.
7.5.8. Other Insect Neuropeptides
Pyrokinin (SGGFAFSPRLamide) and a SIFamide (GYRKPPNGSIFamide) were also
quantified (Fig. 7.13). No significant changes were observed, but the overall trend of an
immediate increase followed by an overshoot of the baseline and/or return to baseline also holds
for these two peptides. Both have been studied in detail in insects. Pyrokinins are primarily
regulators of reproductive function in insects, and they were the only NP that decreased in the
brain of C. borealis following feeding [14]. It is thus likely that pyrokinins are released,
potentially from neurons with cell bodies in the brain, into the hemolymph following feeding. As
most NPs previously discussed seem to act as signals from other parts of the body to the brain,
the presence of an NP that potentially signals in the other direction is of note. SIFamide is
released by midgut epithelial endocrine cells after their depolarization [27] and activates the
pyloric rhythm [28]. It was not significantly changed in the brain after feeding, so the site of
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action for SIFamide may be outside of the brain. These results suggest a possible role for
SIFamide as a communicator from the midgut to organs of the stomatogastric nervous system, to
increase stomach contractions.
7.6. Conclusions and Future Directions
The quantities of 28 neuropeptides in the hemolymph of the crab Cancer borealis were
monitored in 30-min time segments following feeding using microdialysis and novel MS/MS
tools. The measures of changes in NP profiles were determined to be statistically relevant and
not caused by multiple repeated sampling. Of these NPs, 5 were observed to change significantly
at times after feeding. Most NPs followed a pattern of release, overshoot, and recovery within a 3
hr time period after feeding. Orcokinins and AST-B do not appear to change greatly following
feeding. Proctolin is released more gradually and does not return to baseline, even after 3 hrs.
Slight differences in NPs from the same families may point to different roles, or sequential
release to sustain the signal even in the presence of receptor desensitization.
The RFamides were the most significantly altered family of NPs, with 4 changed
significantly. They appear to be involved in the active phases of feeding behavior and
GAHKNYKRFamide levels may even mirror two distinct episodes of feeding that are frequently
observed in the crab; one immediately after the food is present, followed by a short lull in
feeding activity that precedes a second feeding period. This pattern is not statistically significant
and feeding behavior in crabs has not been described quantitatively, nor have the physiological
changes during feeding been determined. For instance, is the two-stage feeding behavior related
to filling the stomach with food, followed by a period of digestion that removes some ingested
material out of the stomach to make room for more food, which is then consumed in the second
feeding period? If so, it is possible that this peptide is involved in the initiation of feeding
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behavior, or “reminds” the crab to eat again once its stomach has been partially emptied. This is
a potentially important function as it informs the animal not only about the state of its stomach
(empty or full) but indicates to the crab that more food than can fit in the stomach is desired.
From experiments in which blue crabs, Callinectes sapidus, were allowed to eat ad libitum for an
hour and then dissected, food was present in the stomach during dissection (Chapter 6). Further
investigation of stomach filling and emptying in the crab would be of use for interpretation of the
data obtained here.
The other RFamides and a YRamide that had significant changes followed the same
pattern as most other NPs mentioned previously. This pattern suggests that release of the peptide
occurs after feeding, but it is removed from circulation rather rapidly, perhaps to tell the animal
to stop eating. This active response indicates that these peptides are likely correlated with the
behavior of feeding itself and are not signals of the body’s energy stores.
This work demonstrates the application of cutting-edge analytical chemistry tools to the
study of NPs in a dynamic physical process, and information potentially relevant to
understanding neuroendocrine control of feeding behavior was obtained. Additional replicates
would improve the quality of the measurements and perhaps increase the number of statistically
significant changes. As it has been demonstrated that the changes here are valid measures,
increasing the number of experimental replicates to 5 or 6 individual crabs may make a great
impact in the significance of these findings. The same data could be processed again, looking for
other NPs. Although 28 were chosen for quantification, an untargeted approach could be done
similar to that described in Chapter 6. Another untargeted approach, that will be described in
Chapter 8, in which all known small- to medium-sized crab NPs (~500) are quantified in some of
the samples to provide a shorter list for quantification in all samples, could also be applied to this
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same dataset. This process is challenging and time-consuming, but could yield new NPs not
previously detected in feeding studies of the crab. It is likely that several important NPs were not
quantified based on the choice to focus on the 28 previously studied; a different MS method was
used in the previous study and it is biased toward better detection of certain types of NPs, and
detects only the most abundant compounds. Finally, the time resolution of the experiment could
be improved by employing affinity-enhanced MD (Chapter 4), using different sample handling
techniques, and/or using a different type of MS detection that requires less sample.
This work provides a useful experimental framework for combining analytical chemistry
techniques into a behavioral neuroscience experiment, and indicates some putative feedingrelated neuropeptides in the crab Cancer borealis. Further refinement of the feeding experiment,
additional replicates, and re-analysis of additional peptides may be useful in providing more
confident assignment of functions for these NPs. Application of this technique to other
behavioral experiments on a similar time scale are likely to be successful in quantifying changes
in NPs for assessment of function.
7.7. Respective Contributions Statement and Acknowledgements
CMS designed and carried out experiments and wrote the paper. LL was responsible for
the initial ideas and theory, and revised the paper.
Heidi L. Behrens is acknowledged for initially developing the crab microdialysis
technique [12, 13] and training. Zhidan Liang is acknowledged for conducting the surgical
implantation of an MD probe for the crab used as biological replicate #3. Kirk J. Grubbs is
acknowledged for assistance in application of statistical methods.
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22. Huybrechts, J.; Nusbaum, M. P.; Bosch, L. V.; Baggerman, G.; Loof, A. D.; Schoofs, L. Neuropeptidomic
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23. Yasuda-Kamatani, Y.; Yasuda, A. Characteristic expression patterns of allatostatin-like peptide, FMRFamiderelated peptide, orcokinin, tachykinin-related peptide, and SIFamide in the olfactory system of crayfish
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MALDI mass spectrometry. J Am Soc Mass Spectrom. 2009, 20, 708-718.
26. Audsley, N.; Weaver, R. J. Neuropeptides associated with the regulation of feeding in insects. General and
Comparative Endocrinology. 2009, 162, 93-104.
27. Christie, A. E.; Kutz-Naber, K. K.; Stemmler, E. A.; Klein, A.; Messinger, D. I.; Goiney, C. C.; Conterato, A. J.;
Bruns, E. A.; Hsu, Y.-W. A.; Li, L.; Dickinson, P. S. Midgut epithelial endocrine cells are a rich source of
the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and
294
GYRKPPFNGSIFamide (Gly1-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer
productus. J Exp Biol. 2007, 210, 699-714.
28. Christie, A. E.; Stemmler, E. A.; Peguero, B.; Messinger, D. I.; Provencher, H. L.; Scheerlinck, P.; Hsu, Y. W.;
Guiney, M. E.; de la Iglesia, H. O.; Dickinson, P. S. Identification, physiological actions, and distribution
of VYRKPPFNGSIFamide (Val1)-SIFamide) in the stomatogastric nervous system of the American lobster
Homarus americanus. J Comp Neurol. 2006, 496, 406-421.
295
7.9. Figures
Figure 7.1. Scatterplot of all data A) before and B) after removal of outliers that represent more
than a 5-fold change from baseline. A total of 22 data points out of 1344, or 1.34%, were
removed. Data points removed likely represent errors in chromatogram integration.
60
A)
50
40
30
20
10
0
0
5
10
15
20
25
5
10
15
20
25
30
B)
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0
30
296
Figure 7.2. Changes in the RFamide peptide GAHKNYLRFamide after feeding. Data are
charted at the time in the middle of their collection period. Values plotted are means and SEM
for 6 experiments on 3 separate crabs. Brackets connect levels significantly different from each
other by a Tukey-Kramer test, and the p-values are indicated. This peptide changes significantly
at several time points after feeding and is likely to be a very important feeding-related peptide.
GAHKNYLRFamide
p=0.0497
1.600
p=0.0314
1.400
Fold-Change
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
p=0.0285
p=0.0068
135
165
297
Figure 7.3. Changes in the RFamide peptide SENRNFLRFamide after feeding. Data are charted
at the time in the middle of their collection period. Values plotted are means and SEM for 6
experiments on 3 separate crabs. Brackets connect levels significantly different from each other
by a Tukey-Kramer test, and the p-values are indicated. This peptide has a very large increase
after feeding followed by a return to baseline around 2 hrs later.
SENRNFLRFamide
1.800
1.600
p=0.0208
1.400
Fold-Change
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
p=0.0234
135
165
298
Figure 7.4. Changes in the RFamide peptide GPRNFLRFamide after feeding. Data are charted at
the time in the middle of their collection period. Values plotted are means and SEM for 6
experiments on 3 separate crabs. Brackets connect levels significantly different from each other
by a Tukey-Kramer test, and the p-value is indicated. This peptide increases after feeding and
returns to baseline levels around 2hrs later.
2.000
GPRNFLRFamide
1.800
1.600
Fold-Change
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time after Feeding (min)
p=0.0404
135
165
299
Figure 7.5. Changes in the RFamide peptide APQRNFLRFamide after feeding. Data are charted
at the time in the middle of their collection period. Values plotted are means and SEM for 6
experiments on 3 separate crabs. Brackets connect levels significantly different from each other
by a Tukey-Kramer test, and the p-value is indicated. This peptide increases after feeding and
may drop to levels below baseline around 2hrs after feeding.
1.600
APQRNFLRFamide
1.400
Fold-Change
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
p=0.0373
135
165
300
Figure 7.6. Overall trends of non-significantly changed NPs in the RFamide family. The overall
trend for peptides in this family is plotted as a solid line with A) AYNRSFLRFamide,
GNRFLRFamide, and GYSKNYLRFamide, and B) NRNFLRFamide, QDLDHVFLRFamide,
and SMPSLRLRFamide. Peptides are shown on two graphs for clarity. Means and SEMs are
plotted. The overall trend for RFamide peptides is an immediate increase after feeding, followed
by an overshoot, and finally a return to baseline values. These results are not statistically
significant.
A) RFamides-1
2.500
AYNRSFLRFamide
GNRFLRFamide
2.000
GYSKNYLRFamide
Fold-Change
Family
1.500
1.000
0.500
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
301
B) RFamides-2
1.800
QDLDHVFLRFamide
1.600
SMPSLRLRFamide
1.400
Family
1.200
Fold-Change
NRNFLRFamide
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
302
Figure 7.7. Changes in HIGSLYRamide after feeding. Data are charted at the time in the middle
of their collection period. Values plotted are means and SEMs for 6 experiments on 3 separate
crabs. Brackets connect levels significantly different from each other by a Tukey-Kramer test,
and the p-values are indicated. This peptide is significantly increased immediately after feeding
and drops to baseline levels significantly after 2hrs post-feeding.
HIGSLYRamide
3.000
2.500
Fold-Change
2.000
1.500
1.000
0.500
0.000
-45
-15
p=0.0301
15
75
105
Time After Feeding (min)
p=0.0117
135
165
303
Figure 7.8. Overall trends of non-significantly changed NPs in the orcokinin family. The overall
trend for peptides in this family is plotted as a solid line with A) NFDEIDRSGFamide,
NFDEIDRSGFA, NFDEIDRSGFGFA, and NFDEIDRSGFGFV, and B) NFDEIDRSSFGFN,
NFDEIDRSSFGFV, and NFDEIDRTGFGH. Peptides are shown on two graphs for clarity.
Means and SEMs are plotted. The overall trend for orcokinins may be a non-feeding-related
slight oscillation in their hemolymph levels. These results are not statistically significant.
A) Orcokinins-1
NFDEIDRSGFamide
NFDEIDRSGFA
NFDEIDRSGFGFA
1.600
NFDEIDRSGFGFV
1.400
Family
Fold-Change
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
304
B) Orcokinins-2
NFDEIDRSSFGFN
NFDEIDRSSFGFV
1.800
NFDEIDRTGFGFH
1.600
Family
Fold-Change
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
305
Figure 7.9. Overall trends of tachykinin-related peptides (TRPs) APSFLGMRamide (CabTRP
Ia) and TPSFLGMRamide (CabTRP II). Means and SEMs are plotted. Other abbreviations are in
the text. Tachykinins appear to increase after feeding, with CabTRP Ia increasing more slowly
than CabTRP II. They then return to baseline. These results are not statistically significant.
Tachykinins
APSGFLGMRamide
2.500
TPSGFLGMRamide
Fold-Change
2.000
1.500
1.000
0.500
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
306
Figure 7.10. Overall trends of RYamides FVGGSRYamide and SGFYANRYamide. Means and
SEMs are plotted. They appear to increase after feeding with a more gradual return to baseline
than some other peptides. These results are not statistically significant.
RYamides
FVGGSRYamide
2.500
SGFYANRYamide
Fold-Change
2.000
1.500
1.000
0.500
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
307
Figure
7.11.
Overall
trends
of
B-type
allatostatins
STNWSSLRSAWamide
and
VPNDWAHFRGSWamide. Means and SEMs are plotted. Slight increases may be observed after
feeding, or oscillations in their hemolymph concentrations that are unrelated to feeding may be
occurring. These results are not statistically significant.
B-Type Allatostatins
1.800
STNWSSLRSAWamide
1.600
VPNDWAHFRGSWamide
1.400
Fold-Change
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
308
Figure 7.12. Overall trends of crustacean cardioactive peptide (CCAP, PFC(-H)NAFTGC(H)amide, (-H) indicates a Cys in a disulfide bond) and proctolin (RYLPT). Means and SEMs are
plotted. CCAP follows a trend similar to many other peptides with changes in their profiles
throughout feeding. Proctolin increases after feeding and continues to stay elevated up to 3hrs
after feeding. These results are not statistically significant.
Other Crustacean NPs
2.500
PFC(-H)NAFTGC(-H)amide
RYLPT
Fold-Change
2.000
1.500
1.000
0.500
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
309
Figure 7.13. Overall trends of a pyrokinin (SGGFAFSPRLamide) and a SIFamide
(GYRKPPNGSIFamide). Means and SEMs are plotted. These peptides again follow the trend of
increase after feeding, followed by a return to baseline. These results are not statistically
significant.
Other Insect NPs
2.000
SGGFAFSPRLamide
1.800
GYRKPPNGSIFamide
1.600
Fold-Change
1.400
1.200
1.000
0.800
0.600
0.400
0.200
0.000
-45
-15
15
45
75
105
Time After Feeding (min)
135
165
310
7.10. Tables
Table 7.1. Peptides quantified in this study and criteria for inclusion of their data in this report.
An ANOVA test and Tukey-Kramer HSD test were conducted to determine if each time point
was significantly different from any of the others at α=0.05. p-Values <0.05 were considered
significant. The bottom row lists the total number of families or peptides in that category.
Family
Peptide Quantified
RFamide
RFamide
RYamide
RFamide
RFamide
RFamide
SIFamide
RFamide
YRamide
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
RFamide
CCAP
RFamide
Proctolin
RFamide
RYamide
Pyrokinin
RFamide
AST-B
TRP
TRP
AST-B
9
APQRNFLRFamide
AYNRSFLRFamide
FVGGSRYamide
GAHKNYLRFamide
GNRFLRFamide
GPRNFLRFamide
GYRKPPNGSIFamide
GYSKNYLRFamide
HIGSLYRamide
NFDEIDRSGFamide
NFDEIDRSGFA
NFDEIDRSGFGFA
NFDEIDRSGFGFV
NFDEIDRSSFGFN
NFDEIDRSSFGFV
NFDEIDRTGFGFH
NRNFLRFamide
PFC(-H)NAFTGC(-H)amide
QDLDHVFLRFamide
RYLPT
SENRNFLRFamide
SGFYANRYamide
SGGFAFSPRLamide
SMPSLRLRFamide
STNWSSLRSAWamide
APSGFLGMRamide
TPSGFLGMRamide
VPNDWAHFRGSWamide
28
Significantly
Different
x
x
x
x
x
5
311
Table 7.2. Complete summary of data for this chapter. At each time point for each peptide, the
average fold-change, standard error, and number of observations are reported. Abbreviations in
text.
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Proctolin
Pyrokinin
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RYamide
RYamide
SIFamide
Tachykinin
YRamide
AST-B
AST-B
CCAP
Table 7.2
Family
STNWSSLRSAWamide
VPNDWAHFRGSWamide
PFC(-H)NAFTGC(H)amide
NFDEIDRSGFamide
NFDEIDRSGFA
NFDEIDRSGFGFA
NFDEIDRSGFGFV
NFDEIDRSSFGFN
NFDEIDRSSFGFV
NFDEIDRTGFGFH
RYLPT
SGGFAFSPRLamide
APQRNFLRFamide
APSGFLGMRamide
AYNRSFLRFamide
GAHKNYLRFamide
GNRFLRFamide
GPRNFLRFamide
GYSKNYLRFamide
NRNFLRFamide
QDLDHVFLRFamide
SENRNFLRFamide
SMPSLRLRFamide
FVGGSRYamide
SGFYANRYamide
GYRKPPNGSIFamide
TPSGFLGMRamide
HIGSLYRamide
Time Point (min)
Peptide
1.017
0.948
0.879
0.752
0.916
0.913
1.003
0.965
0.926
0.960
0.954
0.947
0.990
0.942
1.017
0.932
0.964
0.948
0.960
0.974
0.986
0.899
1.059
0.925
0.954
1.017
0.963
0.941
Average
0.049
0.042
0.069
0.106
0.038
0.053
0.022
0.054
0.043
0.042
0.050
0.034
0.038
0.013
0.020
0.025
0.024
0.034
0.026
0.015
0.028
0.021
0.120
0.024
0.053
0.026
0.066
0.051
-45
Standard
Error
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
n
0.983
1.052
1.121
1.248
1.084
1.087
0.997
1.035
1.074
1.040
1.046
1.053
1.010
1.058
0.983
1.068
1.036
1.052
1.040
1.026
1.014
1.101
0.941
1.075
1.046
0.983
1.037
1.059
Average
0.049
0.042
0.069
0.106
0.038
0.053
0.022
0.054
0.043
0.042
0.050
0.034
0.038
0.013
0.020
0.025
0.024
0.034
0.026
0.015
0.028
0.021
0.120
0.024
0.053
0.026
0.066
0.051
-15
Standard
Error
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
n
1.084
1.326
1.228
1.215
1.399
1.254
0.964
1.487
1.507
1.352
1.113
1.189
1.265
1.369
1.612
1.620
1.223
1.358
1.183
1.312
1.634
1.558
1.470
1.498
2.051
0.946
1.272
1.412
Average
0.124
0.138
0.193
0.227
0.154
0.149
0.055
0.296
0.318
0.235
0.115
0.128
0.111
0.133
0.438
0.474
0.112
0.178
0.101
0.247
0.524
0.350
0.350
0.433
0.531
0.152
0.130
0.176
15
Standard
Error
5
6
6
6
6
6
6
5
6
6
5
6
6
6
6
5
6
6
6
6
5
6
5
5
5
6
6
6
n
1.014
1.068
1.105
1.126
0.982
0.814
0.779
1.214
1.454
0.954
1.474
0.926
1.067
1.023
1.027
1.375
1.159
1.061
0.906
1.090
1.135
1.467
1.180
1.203
0.993
1.174
0.999
1.038
Average
0.106
0.084
0.207
0.171
0.136
0.075
0.059
0.226
0.174
0.056
0.471
0.063
0.040
0.060
0.105
0.272
0.217
0.103
0.061
0.070
0.129
0.353
0.140
0.267
0.164
0.383
0.075
0.089
45
Standard
Error
6
6
6
6
6
6
6
5
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
5
6
6
5
n
312
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Proctolin
Pyrokinin
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RFamide
RYamide
RYamide
SIFamide
Tachykinin
YRamide
AST-B
AST-B
CCAP
Family
STNWSSLRSAWamide
VPNDWAHFRGSWamide
PFC(-H)NAFTGC(H)amide
NFDEIDRSGFamide
NFDEIDRSGFA
NFDEIDRSGFGFA
NFDEIDRSGFGFV
NFDEIDRSSFGFN
NFDEIDRSSFGFV
NFDEIDRTGFGFH
RYLPT
SGGFAFSPRLamide
APQRNFLRFamide
APSGFLGMRamide
AYNRSFLRFamide
GAHKNYLRFamide
GNRFLRFamide
GPRNFLRFamide
GYSKNYLRFamide
NRNFLRFamide
QDLDHVFLRFamide
SENRNFLRFamide
SMPSLRLRFamide
FVGGSRYamide
SGFYANRYamide
GYRKPPNGSIFamide
TPSGFLGMRamide
HIGSLYRamide
Time Point (min)
Peptide
0.922
0.975
1.129
1.223
1.131
0.988
0.856
1.330
1.284
0.861
0.937
1.056
1.203
1.144
1.027
1.096
1.000
1.218
0.948
1.028
1.163
1.101
1.085
0.954
1.020
0.900
1.028
1.422
Average
0.110
0.055
0.190
0.226
0.107
0.133
0.055
0.273
0.197
0.133
0.109
0.061
0.088
0.068
0.055
0.036
0.078
0.092
0.062
0.075
0.107
0.055
0.200
0.059
0.120
0.110
0.064
0.353
75
Standard
Error
6
6
6
6
6
6
6
4
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
n
0.928
0.949
1.024
1.008
0.955
0.794
0.762
1.797
1.058
0.801
1.285
0.959
0.894
1.008
0.902
1.066
0.896
1.095
0.838
0.882
0.985
1.085
1.093
0.937
1.215
0.825
0.962
1.146
Average
0.122
0.066
0.172
0.172
0.126
0.090
0.063
0.501
0.145
0.063
0.413
0.081
0.060
0.041
0.129
0.155
0.094
0.118
0.074
0.105
0.092
0.295
0.214
0.049
0.280
0.177
0.111
0.368
105
Standard
Error
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
n
1.082
0.998
1.008
1.112
1.019
0.990
1.023
1.519
1.177
0.845
0.961
1.046
0.837
1.155
0.813
1.051
0.884
1.061
0.842
0.993
1.207
0.949
1.000
1.009
0.838
0.867
1.099
1.132
Average
0.174
0.100
0.130
0.177
0.091
0.155
0.175
0.317
0.181
0.063
0.135
0.133
0.112
0.179
0.062
0.161
0.069
0.102
0.065
0.146
0.217
0.128
0.139
0.071
0.163
0.115
0.105
0.276
135
Standard
Error
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
n
0.971
1.039
0.991
1.018
0.947
0.817
0.863
1.688
1.337
0.997
1.082
1.028
0.918
1.263
0.931
1.080
0.874
1.029
0.903
1.026
1.005
1.133
1.051
0.931
1.069
0.943
0.986
1.174
Average
0.188
0.150
0.183
0.185
0.166
0.110
0.084
0.407
0.303
0.151
0.234
0.106
0.079
0.198
0.098
0.159
0.047
0.077
0.096
0.131
0.162
0.166
0.177
0.083
0.164
0.137
0.136
0.431
165
Standard
Error
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
6
6
n
313
314
Table 7.3. Summary of statistics for GAHKYLRFamide. Significant (p<0.05) values obtained
from an ANOVA followed by Tukey-Kramer post-hoc analysis are enumerated. In A), time
points that are not connected by a letter are significantly different. Standard error (SE) of the
difference and the confidence levels (CL) at α=0.05 are indicated.
GAHKNYLRFamide
A) Values that are Different
Time
15
A
75
A
45
A
-15
A
-45
A
165
105
135
B
B
B
B
B
B
C
C
C
C
C
C
Mean
1.27
1.20
1.07
1.01
0.990
0.918
0.894
0.837
B) Statistics for Pairs of Different Values
Time Compared to Time Difference SE Difference Lower CL Upper CL p-Value
15
135
0.428
0.108
0.0814
0.774
0.006
15
105
0.370
0.108
0.0243
0.717
0.0285
75
135
0.366
0.108
0.0202
0.713
0.0314
15
165
0.346
0.108
0.000242
0.693
0.0497
Table 7.4. Summary of statistics for SENRNFLRFamide. Significant (p<0.05) values obtained
from an ANOVA followed by Tukey-Kramer post-hoc analysis are enumerated. In A), time
points that are not connected by a letter are significantly different. Standard error (SE) of the
difference and the confidence levels (CL) at α=0.05 are indicated.
SENRNFLRFamide
A) Values that are Different
Time
15
-15
-45
75
A
A
A
A
B
B
B
Mean
1.18
1.04
0.960
0.948
315
45
A
B
0.906
165
A
B
0.903
135
B
0.842
105
B
0.838
B) Statistics for Pairs of Different Values
Compared to
SE
Time
Difference
Time
Difference
15
105
0.345
0.0975
15
135
0.341
0.0975
Lower
CL
0.0337
0.0292
Upper
CL
0.657
0.652
pValue
0.0208
0.0234
Table 7.5. Summary of statistics for GPRNFLRFamide. Significant (p<0.05) values obtained
from an ANOVA followed by Tukey-Kramer post-hoc analysis are enumerated. In A), time
points that are not connected by a letter are significantly different. Standard error (SE) of the
difference and the confidence levels (CL) at α=0.05 are indicated.
GPRNFLRFamide
A) Values that are Different
Time
Mean
15
A
1.61
45
A
B
1.03
75
A
B
1.03
-45
A
B
1.02
-15
A
B
0.983
165
A
B
0.931
105
A
B
0.902
135
B
0.813
B) Statistics for Pairs of Different Values
Time Compared to Time Difference SE Difference Lower CL Upper CL p-Value
15
135
0.798683
0.243
0.0212
1.58
0.0404
Table 7.6. Summary of statistics for APQRNFLRFamide. Significant (p<0.05) values obtained
from an ANOVA followed by Tukey-Kramer post-hoc analysis are enumerated. In A), time
points that are not connected by a letter are significantly different. Standard error (SE) of the
difference and the confidence levels (CL) at α=0.05 are indicated.
316
APQRNFLRFamide
A) Values that are Different
Time
15
A
-15
A
B
165
A
B
-45
A
B
45
A
B
75
A
B
135
A
B
105
B
B) Statistics for Pairs of Different Values
Time
15
Compared to Time
105
Difference
0.550
Mean
1.35
1.04
0.997
0.960
0.954
0.861
0.845
0.801
SE Difference
0.166
Lower CL
0.0197
Upper CL
1.08
p-Value
0.0373
Table 7.7. Summary of statistics for HIGSLYRamide. Significant (p<0.05) values obtained from
an ANOVA followed by Tukey-Kramer post-hoc analysis are enumerated. In A), time points that
are not connected by a letter are significantly different. Standard error (SE) of the difference and
the confidence levels (CL) at α=0.05 are indicated.
HIGSLYRamide
A) Values that are Different
Time
15
A
105
A
165
A
-15
A
75
A
45
A
-45
135
B
B
B
B
B
B
B
Mean
2.05
1.21
1.07
1.05
1.02
0.993
0.953
0.838
B) Statistics for Pairs of Different Values
Time
15
15
Compared
to Time
135
-45
Difference
1.21
1.10
SE
Difference
0.322
0.322
Lower
CL
0.182
0.0659
Upper CL
p-Value
2.244
2.13
0.0117
0.0301
317
Chapter 8: Quantification of Neuropeptides Altered in Acute and Repeated Ethanol
Exposure
Adapted from Schmerberg, C. M., Hayes, K. T., Putterman, L. B., and Li, L. [in preparation]
8.1. Abstract
The role of neuropeptides (NPs) in motivated behaviors relevant to a variety of
pathologies of the reward system is currently under a great deal of investigation [1-7]. One
method to understand the pathology of these disorders is to characterize long-term changes in the
neural substrates of the reward system after exposure to drugs of abuse [8]. The decapod
crustacean is a novel animal model for studying NP changes after long-term drug exposure, as it
expresses a variety of NPs that are homologous to some of those NPs postulated to be involved
in motivated behaviors in mammals [9], and a wealth of analytical tools exist to study these
compounds with high specificity [10]. Recently, evidence has also been presented describing
drug-related conditioning in the crayfish Orconectes rusticus [11-15], and studies linking
neuropeptide F in arthropods to reproduction-related reward [16]. In this chapter, highspecificity, information-rich tandem mass spectrometry (MS/MS) with data-independent
acquisition (DIA) is conducted to quantify changes in the NP content of a major crustacean
neuroendocrine organ, the pericardial organ (PO), of the rock crab, Cancer irroratus, after acute
and repeated ethanol exposure. Isotopic formaldehyde labeling [17-19] was employed in duplex
and triplex schemes in conjunction with DIA MS/MS for quantification. All known crustacean
NPs of an appropriate size (approximately 475 NPs) were quantified after repeated ethanol
exposure, and 77 changed significantly from control. Of these, large (>25%) changes were
observed in 22, all of which were increased. This list of NPs was then used to quantify NP
changes after short-term exposure at three different doses of ethanol, and fold-changes from
318
control were calculated. All were increased in short-term exposures for the same dose of ethanol
that was used in the repeated exposures, while most were decreased or unchanged at lower and
higher doses. An assay to determine the absorption efficiency of ethanol in crustaceans through
immersion based on hemolymph alcohol content was also developed and applied. These results
have implications for understanding NP release by the PO upon ethanol exposure, both acute and
repeated, and potentially adaptation to repeated exposure to a human drug of abuse.
8.2. Introduction
It is becoming clear that neuropeptides (NPs) are important chemical messengers in
motivated behaviors, such as feeding and drug-induced reward [1-7]. Disorders in the brain’s
motivation system can lead to a variety of debilitating pathologies, including compulsive
overeating and addiction to drugs, gambling, sex, etc. The unifying hypothesis is that addiction
or compulsive behavior is a disorder by which a behavior or substance alters the brain’s natural
reward mechanisms, such that natural rewards no longer stimulate it sufficiently. A recent review
characterized alcohol addiction as a “reward deficient disorder” with compulsive behavior
caused by activation of brain stress pathways—in part by the NPs corticotropin-releasing factor
(CRF), dynorphin, and neuropeptide Y (NPY)—and reductions in normal reward function [20].
A multitude of NPs have been implicated in a variety of reward-related disorders in
mammals. For compulsive overeating and food reward, ghrelin, orexin/hypocretin, opioids,
leptin, and neuropeptide Y are just a few of the NPs postulated to be involved [3-7, 21-23].
Orexins, melanin-concentrating hormone (MCH), brain-derived neurotrophic factor (BDNF),
agouti-related protein (AgRP), urocortins, and the previously mentioned CRF, NPY, and
dynorphin, are all implicated in addiction [1, 2, 6-8, 20, 23, 24]. A large and growing body of
319
evidence exists to support the role of NPs as important mediators of reward, both food- and drugrelated, in mammalian systems.
Many of the NPs postulated to be critical in the reward system have a degree of
conservation among mammals and arthropods, including but not limited to peptides related to
oxytocin/vasopressin,
NPY
(neuropeptide
F
is
the
invertebrate
homolog),
and
gastrin/cholecystokinin (CCK) [9]. A recent study has demonstrated increased ethanol drinking
in Drosophila when reproduction-related reward was prohibited, along with a decrease in NPY
brain immunoreactivity [16]. A crustacean model of drug-related reward in the crayfish
Orconectes rusticus has also recently been described [11-15], and other arthropod species have
recently been established as models of drug addiction [25, 26].
One limitation of most previous studies of NPs involved in reward is reliance on
antibody-based techniques for NP detection. As discussed in previous chapters, this method
suffers from antibody-cross reactivity and is only able to provide limited information. A wealth
of high specificity tools for studying NP changes in the decapod crustacean based on mass
spectrometry (MS) and tandem MS (MS/MS) have been developed for improved selectivity of
NP identification and the potential to obtain sequence information directly from small samples
[10]. A highly specific method of quantifying NPs and verifying their sequences using the newly
developed MS/MS acquisition method, data-independent acquisition (DIA), has been applied
previously in this dissertation to quantify NPs changed in feeding with high specificity and
accuracy in the decapod crustacean (Chapter 7).
Although it is currently unknown if alcohol exposure is a rewarding experience for
crustaceans, other drugs that are rewarding in humans are also rewarding in crayfish [14, 15] and
most drugs of abuse act on common neural pathways in mammals to produce reward. It is
320
currently unclear which of alcohol’s many effects is responsible for its reinforcing properties. In
the classical model, ethanol increases dopamine in the mesolimbic dopamine system by
inhibition of GABA-ergic neurons that tonically inhibit dopaminergic neurons in the ventral
tegmental area (VTA) [27]. This is by no means sufficient to explain the complex mechanisms
by which alcohol is rewarding, however. Alcohol also is thought to alter levels of endogenous
opioids for positive reinforcement and/or cessation of negative reinforcement through currently
unknown mechanisms [28]. In the crustacean, GABA is also an inhibitory neurotransmitter in the
central and peripheral nervous systems, and further activation of GABA receptors by ethanol
may increase inhibitory transmission.
In this chapter, long-term changes in the NP complement of a major neuroendocrine
organ, the pericardial organ (PO), of the Rock crab Cancer irroratus after repeated exposure to a
human drug of abuse, ethanol, are determined and compared to short-term exposure changes.
The advantage of this approach is that all known putative NPs can be quantified, and the data can
be re-analyzed in the future as additional information becomes available. Isotopic formaldehyde
labeling [17-19] was also employed in duplex and triplex schemes in conjunction with DIA
MS/MS quantitation for the first time. Using an untargeted quantitation approach, approximately
475 NPs were quantified after repeated daily exposure to 0.2 M ethanol for 1 hr. Of these NPs,
77 changed significantly from controls, and 22 of these changed more than 25%, all of which
were increased compared to controls. These were quantified in short-term 1hr exposure samples
from crabs exposed to three different doses of ethanol (0.125 M, 0.2 M, and 0.5 M), and foldchanges from control were calculated. All were increased in short-term exposures to 0.2 M
ethanol, while most were decreased or unchanged after 0.125 M and 0.5 M exposure. Twelve
increases in the 0.2M dose were statistically significant compared to control animals, and no
321
statistical difference was observed at the other doses. The relevance of this dose-dependency is
currently unknown. An assay to determine the extent to which ethanol is absorbed by the crab
through the gills when immersed by quantifying hemolymph levels is also developed.
Interestingly, after short-term exposure, the hemolymph alcohol levels were the same for crabs
exposed to 0.2M or 0.5M ethanol. These results have implications for understanding NP release
by the PO upon ethanol exposure, both acute and chronic, and potentially adaptation to repeated
exposure to a human drug of abuse.
8.3. Methods
Several of the methods have been described previously in Chapters 4, 6, and 7, and are
presented in detail in Appendix A. Only where the methods differ from previous chapters will
they be discussed here.
8.3.1. Materials
Unless otherwise indicated, reagents were purchased from Fisher Scientific (Waltham,
MA). Alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, ≥300 units/mg protein
(product # A3263 Sigma Aldrich, St. Louis, MO), phenazine methosulfate (PMS, Sigma Adrich),
β-nicotinamide adenine dinucleotide hydrate (NAD+, Sigma Aldrich), thiazolyl blue tetrazolium
bromide (MTT, Sigma Aldrich), ethanol 100% (Sigma Aldrich), 1mL plastic syringe with ½” 26
ga needle (Becton Dickinson, Franklin Lakes, NJ), crab saline (440 mM NaCl, 26 mM MgCl 2,
13 mM CaCl2, 11.2 mM Trizma base, 11 mM KCl, 5.1 mM maleic acid, pH adjusted to 7.4),
phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM NaH2PO4, 1.76 mM
KH2PO4, pH 7.4), tris buffer (0.1 M Tris-HCl, 0.1% (w/v) Triton X-100, pH 8.5), sodium
dodecyl sulfate (SDS, Sigma-Aldrich), dimethylformamide (DMF, Sigma-Aldrich), methanol,
322
formic acid, 200 proof ethanol (Decon Laboratories, Inc., Bryn Mawr, PA), pellet pestles (Fisher
Scientific), sonicator, centrifuge
8.3.2. Animals
Male rock crabs (Cancer irroratus) were obtained from Ocean Resources (Sedgwick,
ME) and housed in an aquarium system with sump filtration and a protein skimmer. All tanks
had crushed gravel as substrate and water quality was monitored regularly. Regular water
changes were conducted and animals were fed if they were in the tanks for more than 1 week.
Animals were fed raw white fish (purchased from a grocery store) and were allowed to eat for
several hours. The room had temperature, humidity, and light controls. The tanks were
maintained at a water temperature of 10-15°C, and salinity was maintained in the 32 to 38 ppt
range. The room was on a 12-hr reverse light schedule, with lights off from 10 AM to 10 PM—in
order to observe animals during their active period (dark) during normal work hours—and on
from 10 PM to 10 AM.
For dissection, a previously published procedure was followed [29]. Briefly, animals
were put on ice for at least 15min to anesthetize them, and were weighed following anesthesia
immediately prior to dissection. Sinus glands, brains, and pericardial organs were isolated and
stored in tubes containing 50 µL of acidified methanol (90:9:1 MeOH: H2O: acetic acid) on ice.
Tissues were kept in -80°C until extraction.
8.3.3. Ethanol Exposure
All ethanol exposures were conducted in shoe box-sized solid plastic mouse cages with
lids that contained 4L of liquid. Solutions of ethanol (Decon Laboratories) at 0.5, 0.2, and 0.125
M in water from the crabs’ home aquaria were made in these cages. Control animals were placed
in pure aquarium water. For short-term exposure control, 8 crabs were used. For the short-term
323
exposure dosed, the following numbers of animals were used: 0.125M: 4 crabs, 0.2M: 8 crabs,
and 0.5M: 3 crabs. For long-term exposure, two cohorts were used, one with 3 crabs in control
exposure and 3 dosed, and the other with 4 crabs under the control condition and 4 dosed. Prior
to exposure, 0.75mL of hemolymph was withdrawn, and then replaced with an equal volume of
crab saline, with exceptions as noted. Crabs were placed in the cages and left undisturbed for
1hr. At this point, they were removed from the tank, 0.75mL of hemolymph was withdrawn, and
they were either put on ice for dissection or rinsed by immersion in ethanol-free aquarium water
and returned to their home tank. Exposed and control crabs were kept separate with a plexiglass
divider. For long-term studies, 0.75 mL of hemolymph was only taken every other day to avoid
undue stress on the animal; on the other days, 0.2 mL was taken and this volume was not
replaced with crab saline. For long-term exposure studies, a system using colored plastic cable
ties was developed to identify individuals. They were exposed to 0.2 M ethanol for 1hr daily for
5 consecutive days, with dissection occurring immediately after the last exposure. Crabs were
fed on the day prior to the first exposure.
8.3.4. Sample Preparation
The pericardial organs were extracted following a published procedure [18]. Briefly,
tissues were ground in 100 µL acidified methanol, sonicated, and pelleted by centrifugation for 5
min at 16,100 x g. The supernatant was saved and the pellet was re-extracted two additional
times. Supernatants were combined and solvent was evaporated (Savant SC10 Speedvac,
Thermo Scientific). Samples were resuspended and desalted using C18 Ziptips (EMD Millipore,
Billerica, MA). This was dried again and resuspended in 75 µL of 10 nM bradykinin (American
Peptide, Sunnyvale, CA) internal standard solution. Formaldehyde labeling was conducted
following one of two published procedures [18] or [17]. For long-term samples, a two-plex
324
dimethyl labeling strategy employing formaldehyde and deuterium formaldehyde with borane
pyridine as hydride source was employed [18]. For short-term samples, a three-plex dimethyl
labeling adding
13
C deuterium formaldehyde was employed, using NaCNBH3 or NaCNBD3 as
the hydride source [17]. Samples were assigned to groups based on crab size. A control sample
was present in each group. The assignment of light, intermediate, or heavy label was conducted
randomly to blind the samples for analysis and eliminate any labeling bias. After labeling,
samples were evaporated to dryness, resuspended in 10 µL 0.1% FA, and mixed.
8.3.5. Instrumental Analysis
Samples were placed in LC vials and analyzed as in Chapters 6 and 7 and Protocol A.
Ultrahigh performance liquid chromatography (UPLC)-MSE analysis was conducted on a Waters
nanoAcquity UPLC coupled to a Waters Synapt G2 mass spectrometer (Waters, Milford, MA).
Samples (2 µL) were trapped on a preconcentration column and desalted on-line. This column
was then put in line with a C18 reversed-phase column (BEH130 C18, 1.7 µm, 75 µm x 100 mm,
Waters, Milford, MA). The outlet of this column was connected to a fused silica capillary with a
pulled tip of internal diameter ~5 µm (Sutter Instrument Company, Novato, CA). This was used
as the ESI inlet. A 75 min reversed-phase run with solvent A as 0.1% FA in water and B as 0.1%
FA in ACN was used. The instrument was operated in data-independent MS/MS mode with the
high energy scan having a voltage ramp from 25 to 65 V and Glu-fibrinopeptide was infused for
lockspray calibration.
8.3.6. Data Analysis
A previously developed pseudo multiple reaction monitoring (pMRM) method for
quantification of MSE data (Chapters 6, 7) using Skyline software (University of Washington
[30]) was developed for use in this chapter. A list of all known crustacean neuropeptides (~475
325
NPs) smaller than 1500 Da and excluding the CHH-precursor related peptides was created for
data extraction and integration of long-term exposure samples. A shorter list containing 22
peptides was created from the long-term exposure results for analysis of the short-term exposure
data in a method described below. The internal standard bradykinin was added to each list, and
settings for triplex dimethylation were input into the program. The iRT function with retention
time predictor was used, calibrated off of results from Mascot (Matrix Science, London, UK)
identifications of equine myoglobin tryptic peptides in a previous LC run of the myoglobin
digest alone under the same UPLC conditions. The 2+ and 3+ precursors were monitored to the
respective transitions to their most abundant 1+ and 2+ b and y ions. MS/MS filtering parameters
were set to DIA with MSe isolation, and 10,000 resolving power. Peak integration was checked
manually for a good match to the most abundant transitions and agreement with the predicted
retention times. Variation was greater between sets of samples run on the instrument on different
days than within a single day. All long-term samples were subjected to UPLC-MS/MS on the
same day and analyzed in parallel. Similarly, all short-term samples were injected on UPLCMS/MS on a different day but also analyzed in parallel. Peak areas were exported to Microsoft
Excel.
All transitions for each isotopic form of the labeled peptide were summed. These were
normalized to the corresponding isotopic-labeled signal for bradykinin and divided by the body
weight of the crab. Conditions were unblinded, and the mean and variance was calculated for
each isotope-labeled peptide. The average variance was also conducted, and samples whose
variance was greater than 3 times the average for that peptide were considered outliers and were
removed from analysis. A minimum of 2 outliers from 7 samples were observed, and most had 1
or no outliers. A student’s paired t-test was conducted, and significance was set at p<0.05. The
326
ratio of dosed to control was determined for each peptide. For those peptides with a significant
difference according to the t-test and a ratio greater than 1.25 or less than 0.75, detailed results
are shown. These 22 peptides were used to create the list for analysis of the short-term exposure
samples. For short-term samples, all internal standard- and crab weight-normalized values were
compared to control values using a student’s t-test with significance threshold at p<0.05. The
values were then divided by the control value to yield fold-change.
8.3.7. Alcohol Dehydrogenase (ADH) Assay
Hemolymph was collected from the crabs as described previously. Additional
hemolymph from an un-dosed crab was used to make blanks and calibration curve samples.
Hemolymph was put on ice immediately after collection and spun down at 16,100 x g for 5
minutes to get crab plasma. Blank plasma can be stored in the freezer for several weeks. A
calibration curve consisting of 10, 5, 3, 1, and 0.1 mM EtOH (Sigma Aldrich) in crab plasma was
created. To each well of the microplate (96-well Perkin Elmer Isoplate, white frame and clear
well, Perkin Elmer, Waltham, MA) were added 76.7 µL of Tris buffer (0.1 M Tris-HCl, 0.1%
(w/v) Triton X-100, pH 8.5), 10 µL of 600 uM MTT (in Tris buffer), and 10 µL of 37.7 mM
NAD+ (in Tris buffer) solution. Samples, calibrants, and blank were then added to each well in
volumes of 4 µL. Samples from crabs exposed to alcohol were also diluted 1:4 with Tris buffer
and 4 µL of this was put in wells. Two µL of 5 pM phenazine methosulfate (PMS) was added
followed by 4 µL of 7.33x106 U/L ADH. The plate was mixed on a rotating platform (Fisher
Scientific Clinical Rotator) for 1 minute and incubated at 50 °C for 60 minutes. The reaction was
stopped by adding 100 µL of inhibitor (20% SDS (w/v) in 50/50 DMF/H2O (v/v)) to each well,
followed by another 1min of mixing. The absorbance at 570 nm and 655 nm was determined
with a Tecan Ultra 384 microplate reader with XFluor4 software (Tecan Group Ltd., Männedorf,
327
Switzerland) and filters from Omega Optical (Omega Optical, Inv., Brattleboro, VT). Data was
analyzed in Microsoft Excel, plotting absorbance at 570nm – absorbance at 655nm against EtOH
concentration. A linear regression was generated and used to calculate the concentrations of
unknown samples within the calibrated range. This assay was developed by modification of
reference assays to crab hemolymph samples [31-33].
8.4. Results and Discussion
This study successfully quantified 475 NPs in samples labeled with H- and Dformaldehyde and analyzed via DIA MS/MS. A large number of these were found to have
statistically significant changes in animals repeatedly exposed to alcohol. Large changes were
observed in 22 of these, which were then quantified in samples labeled with H-formaldehyde, Dformaldehyde, and 13C D-formaldehyde and NaCNBD3. The application of isotopic labeling with
DIA MS/MS quantitation has not previously been reported, and the 3-plex dimethylation scheme
is less frequently used. This work demonstrates that stable isotope labeling and DIA MS/MS can
be combined with pseudo-SRM data analysis for highly accurate quantitation of peptides in
large-scale analysis.
8.4.1. Repeated Alcohol Exposure
Two cohorts of crabs were subjected to daily, 1hr exposures to 0.2 M ethanol (or plain
aquarium water) for 5 consecutive days. NPs from the POs of animals exposed to ethanol had 77
significantly altered NPs, all of which were increased relative to control animals. Large changes
(>25%) were observed for 22 NPs, indicated in Fig. 8.1.A and B. One combination allatostatin
(AST Combo, a prepropeptide sequence containing multiple putative active ASTs), 9 A-type
ASTs (AST-A), 2 B-type ASTs (AST-B), one kinin, one YRamide, 5 RFamide-like peptides, one
RYamide, and 2 tachykinin-related peptides are shown.
328
Increases in NP levels in general after alcohol exposure are not surprising. Due to its
positive effects on GABA inhibitory signaling, alcohol can decrease overall neuronal activity
and thus decrease any constitutive release of NPs by the PO into the hemolymph. However, over
a period of several days, cells might decrease NP synthesis to compensate for a period during
which release was reduced (the daily exposure period). On the other hand, inhibition of overall
neuronal activity is not the only mechanism by which ethanol acts in the mammal, and other
processes may be at work in the crab as well, such as effects on NP release, which ethanol
stimulates in some regions of the brain. It is possible that after 5 days, changes in the synthesis
rate of NPs have not occurred, or that increased levels of NPs are a result of increased synthesis
of NPs starting immediately after immersion in the tank.
Changes in allatostatins are of interest because most of these compounds were not
observed to change significantly following feeding. Their overall activity is to reduce synthesis
of juvenile hormones and decrease neuronal activity (see Ch. 2). This effect is similar to the
classic effect of ethanol through activation of the GABA receptor. It is possible that their
increased levels in the PO are in response to adaptation of the animal to reduced overall neuronal
activity when ethanol is present, and ASTs serve the same purpose after the animal’s exposure is
over, in order to maintain a certain overall level of activity.
The second most affected family of NPs, the RFamide-like peptides, contains family
members that stimulate and inhibit firing of muscles and neurons (See Ch. 2). Most peptides in
this family increase contractions in the heart and other muscles, but not all do. In Ch. 7 it was
observed that RFamides increased in the hemolymph immediately following feeding, and this is
in agreement with tissue extract findings [18]. Their increase in the PO tissue after repeated
alcohol exposure may be due to increased synthesis of these peptides potentially related to
329
neuropeptide Y and CCK. Longer after alcohol exposure, they may be released in large
quantities into the hemolymph, potentially as a reward signal. Alternatively, they may be
released after alcohol exposure to increase the basal tone of active cells to counteract negative
effects of ethanol. Other peptides that were altered are also considered stimulatory peptides, and
this may account for their increase.
8.4.2. Short-Term Alcohol Exposure
For the peptides with large, significant changes after repeated ethanol exposure, large
increases were also observed in the 0.2 M dose after short-term exposure (see Fig. 8.2.), and 12
of these were statistically significant compared to control (p<0.05). For a smaller dose, 0.125 M,
most were decreased or close to unchanged compared to control, and for a larger dose, 0.5 M,
little change from control was observed. Because the profiles look so similar for each peptide, it
was verified statistically that they were not all simply measurements of the same background
signal; values are significantly different between peptides.
The relevance of these findings is currently unclear. When taken with information on the
hemolymph ethanol concentrations (HECs) following short-term exposure (Table 8.1), it appears
that the same HEC is causing different effects in the PO NP content between the 0.2M and 0.5M
doses. The increase in PO levels of these NPs, which was statistically significant compared to
control for 12 compounds, may indicate that ethanol is reducing tonic NP release by the PO in
this condition by reducing overall neuronal activity, through its known augmentation of GABAergic signaling. At the lower dose, the HEC may not be high enough to alter baseline release of
NPs from the PO, and thus this dose is not distinguishable from control. At the high dose, there
is the possibility that NP release is stimulated, along with alterations in water elimination from
the animal, or increases in the efficacy of enzymes that metabolize ethanol. Stimulated NP
330
release occurs after ethanol exposure in some regions of the mammalian brain, so it is possible
that the PO is being stimulated to release NPs despite overall increased inhibitory tone in the
nervous system due to the presence of ethanol. This leads to a PO NP level in the 0.5M condition
that is similar to that observed in control. Since the HEC is similar at the 0.5M dose and the
0.2M dose, it is possible that the animal is eliminating water from its circulation more slowly to
increase its overall hemolymph volume to reduce the effects of such a high dose of ethanol.
Other possible explanations include ethanol modulation of the enzymes responsible for its
metabolism. Finally, the higher numbers of animals at the 0.2M and control conditions (8 in each
condition) compared to the 0.125M and 0.5M conditions (4 and 3 animals, respectively), may be
the reason statistical significance is only observed in the 0.2M dose. Additional measurements of
NP levels and HECs at the 0.125M and 0.5M doses may provide a clearer picture and potentially
statistically significant differences from control values. A variety of additional information about
the effects of different doses of ethanol in the crab is necessary to properly interpret these results.
8.4.3. Alcohol Dehydrogenase Assay
The alcohol dehydrogenase assay generated a linear calibration curve for ethanol in the
concentration range 0.1-10 mM (Fig. 8.3.). A representative graph showing the change in ethanol
concentration for crabs exposed to 0.5 M ethanol is shown in Fig. 8.4. Average values for
hemolymph alcohol concentration for short-term exposures are indicated in Table 8.1. For 0.2
and 0.5 M, the concentration was approximately equal. Daily values for before and after
exposures in the repeated exposure experiment are indicated in Fig. 8.5. The experiment was
conducted in two cohorts, one of 3 control and 3 dosed crabs, and one of 4 control and 4 dosed
crabs. For both acute and repeated exposures, the control condition does not lead to an elevation
in hemolymph ethanol levels, and levels before the test are nearly zero. No large change is
331
observed in the post-exposure alcohol levels from day to day in the repeated exposure test. This
does not indicate that crabs are not developing tolerance, as increased elimination rate is not the
only mechanism by which tolerance can be achieved.
8.5. Conclusions and Future Work
The observation that 22 NPs increased greatly and significantly in crabs repeatedly
exposed to ethanol daily over a 5-day period suggests that these NPs may be being retained in
neurons due to overall reduced activity caused by ethanol’s potentiation of GABA signaling. The
synthesis of these NPs could also be upregulated as a result of repeated ethanol exposure, as
some (such as allatostatins) may be used to maintain overall inhibitory neuronal tone after
ethanol exposure is concluded in a homeostatic mechanism. The levels of these compounds in
other tissues and the hemolymph would be of great interest to determine if the increases in the
PO are due to decreased release or increased synthesis. Twelve of the 22 NPs were also
significantly increased after short-term exposure to the same dose of ethanol, 0.2M. Since shortterm exposure is not likely to cause significant changes in NP synthesis, it is probable that these
NPs increase in PO after alcohol exposure as a result of reduced baseline release. Short-term
exposure at other doses did not provide differences in the levels of these 22 NPs in the PO
compared to control. This could be due to ethanol-stimulated NP release at the 0.5M dose, and
not enough ethanol present at the 0.125M dose to inhibit basal NP release, or it could be an
artifact of having fewer experimental animals in the low-dose conditions. Similar HECs for
short-term exposure to 0.2M and 0.5M ethanol could be due to regulation of water balance in the
animal, ethanol-metabolizing enzymes, or again could be an artifact of too few replicates.
Additional information will be required to better understand the implications of this work,
including more experimental replicates, analysis of other tissues, and following concentration
332
changes of ethanol and NPs in the hemolymph throughout ethanol exposure using microdialysis
sampling. The data obtained thus far can also be mined for significant changes in the short-term
samples in additional NPs that may have different expression profiles. Behavioral analysis may
help determine if ethanol is a rewarding stimulus and if tolerance is occurring. More
investigations into the mechanisms of ethanol uptake and elimination in the crab would also be
of use.
8.6. Respective Contributions and Acknowledgments
CMS designed and carried out the experiments and wrote the paper. KTH helped with
sample preparation. LBP helped in developing and applying the ADH assay. LL advised and
revised the paper. Additional advice on experimental design was provided by Brendan M.
Walker of Washington State University, Pullman, WA. Helpful insight was provided by Chenxi
Jia, Zhidan Liang, and Nicole Woodards.
8.7. Works Cited
1. Marchant, N. J.; Millan, E. Z.; McNally, G. P. The hypothalamus and the neurobiology of drug seeking. Cell Mol
Life Sci. 2012, 69, 581-597.
2. Sprow, G. M.; Thiele, T. E. The neurobiology of binge-like ethanol drinking: Evidence from rodent models.
Physiology & Behavior. 2012, 106, 325-331.
3. Fulton, S. Appetite and reward. Frontiers in Neuroendocrinology. 2010, 31, 85-103.
4. Parker, J. A.; Bloom, S. R. Hypothalamic neuropeptides and the regulation of appetite. Neuropharmacology.
2012, 63, 18-30.
5. Menzies, J. W.; Skibicka, K.; Egecioglu, E.; Leng, G.; Dickson, S. Peripheral Signals Modifying Food Reward.
In: Joost H-G, editor. Appetite Control: Springer Berlin Heidelberg; 2012. p. 131-158.
6. Skibicka, K. P.; Dickson, S. L. Ghrelin and food reward: The story of potential underlying substrates. Peptides.
2011, 32, 2265-2273.
7. Thompson, J. L.; Borgland, S. L. A role for hypocretin/orexin in motivation. Behavioural Brain Research. 2011,
217, 446-453.
8. Briand, L. A.; Blendy, J. A. Molecular and genetic substrates linking stress and addiction. Brain Research. 2010,
1314, 219-234.
9. Grimmelikhuijzen, C. J. P.; Hauser, F. Mini-review: The evolution of neuropeptide signaling. Regulatory
Peptides. 2012, 177, Supplement, S6-S9.
10. Li, L.; Sweedler, J. V. Peptides in the brain: mass spectrometry-based measurement approaches and challenges.
Annu Rev Anal Chem (Palo Alto Calif). 2008, 1, 451-483.
11. Dziopa, L.; Imeh-Nathaniel, A.; Baier, D.; Kiel, M.; Sameera, S.; Brager, A.; Beatriz, V.; Nathaniel, T. I.
Morphine-conditioned cue alters c-Fos protein expression in the brain of crayfish. Brain Research Bulletin.
2011, 85, 385-395.
12. Huber, R. Amines and motivated behaviors: a simpler systems approach to complex behavioral phenomena.
Journal of Comparative Physiology a-Neuroethology Sensory Neural and Behavioral Physiology. 2005,
191, 231-239.
333
13. Nathaniel, T. I.; Huber, R.; Panksepp, J. Repeated cocaine treatments induce distinct locomotor effects in
Crayfish. Brain Research Bulletin. 2012, 87, 328-333.
14. Nathaniel, T. I.; Panksepp, J.; Huber, R. Drug-seeking behavior in an invertebrate system: Evidence of
morphine-induced reward, extinction and reinstatement in crayfish. Behavioural Brain Research. 2009,
197, 331-338.
15. Panksepp, J. B.; Huber, R. Ethological analyses of crayfish behavior: a new invertebrate system for measuring
the rewarding properties of psychostimulants. Behavioural Brain Research. 2004, 153, 171-180.
16. Shohat-Ophir, G.; Kaun, K. R.; Azanchi, R.; Mohammed, H.; Heberlein, U. Sexual deprivation increases ethanol
intake in Drosophila. Science. 2012, 335, 1351-1355.
17. Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J. R. Multiplex peptide stable isotope
dimethyl labeling for quantitative proteomics. Nat Protoc. 2009, 4, 484-494.
18. Chen, R.; Hui, L.; Cape, S. S.; Wang, J.; Li, L. Comparative Neuropeptidomic Analysis of Food Intake via a
Multi-faceted Mass Spectrometric Approach. ACS Chem Neurosci. 2010, 1, 204-214.
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isotope dimethyl labeling in quantitative proteomics. Anal Bioanal Chem. 2012, 404, 991-1009.
20. Koob, G. F. Theoretical frameworks and mechanistic aspects of alcohol addiction: alcohol addiction as a reward
deficit disorder. Curr Top Behav Neurosci. 2013, 13, 3-30.
21. Grosshans, M.; Loeber, S.; Kiefer, F. Implications from addiction research towards the understanding and
treatment of obesity. Addiction Biology. 2011, 16, 189-198.
22. Skibicka, K. P.; Dickson, S. L. Ghrelin and food reward: the story of potential underlying substrates. Peptides.
2011, 32, 2265-2273.
23. Plaza-Zabala, A.; Maldonado, R.; Berrendero, F. The Hypocretin/Orexin System: Implications for Drug Reward
and Relapse. Mol Neurobiol. 2012, 45, 424-439.
24. Ryabinin, A. E.; Tsoory, M. M.; Kozicz, T.; Thiele, T. E.; Neufeld-Cohen, A.; Chen, A.; Lowery-Gionta, E. G.;
Giardino, W. J.; Kaur, S. Urocortins: CRF's siblings and their potential role in anxiety, depression and
alcohol drinking behavior. Alcohol. 2012, 46, 349-357.
25. Awofala, A. A. Genetic approaches to alcohol addiction: gene expression studies and recent candidates from
Drosophila. Invert Neurosci. 2011, 11, 1-7.
26. Kaun, K. R.; Devineni, A. V.; Heberlein, U. Drosophila melanogaster as a model to study drug addiction. Hum
Genet. 2012, 131, 959-975.
27. Kandel, E. R.; Schwartz, J. A.; Jessell, T. M. Principles of Neural Science. 4 ed. New York, NY: McGraw Hill;
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28. Walker, B. M.; Valdez, G. R.; McLaughlin, J. P.; Bakalkin, G. Targeting dynorphin/kappa opioid receptor
systems to treat alcohol abuse and dependence. Alcohol. 2012, 46, 359-370.
29. Ma, M.; Wang, J.; Chen, R.; Li, L. Expanding the Crustacean neuropeptidome using a multifaceted mass
spectrometric approach. J Proteome Res. 2009, 8, 2426-2437.
30. MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.;
Liebler, D. C.; MacCoss, M. J. Skyline: an open source document editor for creating and analyzing targeted
proteomics experiments. Bioinformatics. 2010, 26, 966-968.
31. Lim, H. H.; Buttery, J. E. Determination of ethanol in serum by an enzymatic pms-int colorimetric method.
Clinica Chimica Acta. 1977, 75, 9-12.
32. Zanon, J. P.; Peres, M. F. S.; Gattás, E. A. L. Colorimetric assay of ethanol using alcohol dehydrogenase from
dry baker's yeast. Enzyme and Microbial Technology. 2007, 40, 466-470.
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8.8. Figures
Figure 8.1. Fold-change from baseline in repeated exposure experiments for NPs with
statistically significant changes greater than 25%. Values shown are means ± standard error of
the means (SEM). P values indicated with * or **. A) AST-family peptides. B) Other peptides.
A)
B)
335
Figure 8.2. Fold-change from baseline in acute exposure experiments at 3 different doses for
NPs identified in repeated exposure experiments as potentially of interest. Values shown are
means ± SEM. Statistical significance is indicated with a * (p<0.05). A) AST-family peptides. B)
Other peptides.
A)
336
B)
Figure 8.3. Representative graph showing linearity of ADH assay for ethanol concentration in
crab hemolymph.
0.3
Abs (570-655nm)
0.25
0.2
0.15
0.1
y = 0.027x - 0.0076
R² = 0.9552
0.05
0
0
-0.05
2
4
6
8
Ethanol Concentration (mM)
10
12
337
Figure 8.4. Graph illustrating change in hemolymph ethanol levels after acute exposure to 0.5 M
solution. Values indicated are means ± SEM (n=3). Significance value between before and after
values for ethanol exposed crabs is indicated.
0.5M EtOH Immersion
35
EtOH Conc (mM)
30
25
20
Control
15
p=0.0006
EtOH Exposed
10
5
0
before
after
338
Figure 8.5. Hemolymph ethanol levels for crabs in the repeated exposure experiments. Control
and 0.2 M ethanol-exposed crabs shown. For each day and condition, n=7. Experiment was
conducted in two cohorts, one of 3 control, 3 dosed, and one of 4 control, 4 dosed. Values
indicated are means ± SEM. Negative values are an artifact of normalizing the response to a
blank sample of crab hemolymph, which has a small amount of intrinsic activity in the assay that
Control
Day 1
Day 2
Day 3
Day 4
After
Before
After
Before
After
Before
After
Before
Dosed
After
45
40
35
30
25
20
15
10
5
0
-5
Before
Hemolymph EtOH Concentration
(mM)
is variable from one crab to another.
Day 5
8.9. Table
Table 8.1. Hemolymph alcohol concentrations of dosed crabs after short-term exposure. Means
and standard deviations are indicated for each dose. A small amount of baseline activity in the
assay is observed for undosed animals.
Dose (M)
Control
0.125
0.2
0.5
Hemolymph Alcohol Concentration (mM)
After Exposure
Average
SEM
4.42
1.11
12.7
7.98
31.6
4.24
29.4
0.782
Number of Animals (n)
8
4
8
3
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Chapter 9: Conclusions and Future Directions
9.1 Conclusions
In this dissertation, methods were developed for analysis of neuropeptides (NPs) in the
decapod crustacean based on mass spectrometry (MS) and applied to experiments in feeding and
alcohol exposure. A summary of the types of NPs found in crustaceans, MS-based and traditional
biochemical analysis of these compounds, and functional information was presented in Chapter
2. It was established that crustaceans are an excellent model organism for developing new
analytical chemistry tools for use in neuroscience research and that much remains to be
discovered, particularly in identifying the functions of these compounds. New and emerging
methods for conducting neuropeptide identification and functional assignment studies were
described in Chapter 3. These methods are allowing researchers to conduct untargeted
peptidomics research to ensure that all possible compounds of interest are analyzed, and the
decision of which compounds to analyze is based on the data itself.
A technique to improve NP recovery in microdialysis (MD) sampling termed affinityenhanced MD was developed in Chapter 4. This technique is useful for increasing the
concentration of low-abundance compounds in microdialysates and permits quantitation of
compounds present endogenously below the limit of quantitation for the analytical instrument in
use without concentrating sample and losing temporal resolution. The neuropeptidome of the
crayfish Orconectes rusticus was described in Chapter 5 with two different types of MS/MS
interpretation software. This led to a good understanding of the NPs present in this species and
provided insight on methodology for future experiments of a similar nature. In Chapter 6, a
quantification method using a new form of MS/MS acquisition, data-independent acquisition
(DIA), was developed. This method used a software package commonly used for the most
340
specific form of MS/MS quantitation currently in use, MRM. Its linearity over the peptide
concentration range was observed and it was successfully applied to quantitate NPs in tissue
extracts of the blue crab Callinectes sapidus following feeding.
These tools were applied in later chapters to behavioral neuroscience experiments. First,
microdialysis sampling and DIA MS/MS quantitation were employed to study NPs that change
in the hemolymph during feeding in the crab Cancer borealis. This successfully quantified 28
NPs in two replicates from each of three individual crabs. Several significant changes were
observed, particularly among peptides of the RFamide-like peptide family, suggesting a possible
role for these peptides in feeding. Other peptides had non-significant changes, but most appeared
to follow the same trend—rapid increase in hemolymph followed by overshoot and recovery to
baseline levels within 3hrs—with the exception of two groups of peptides that were unchanged
and one that was released more gradually and did not decrease at the end of the time monitored.
In Chapter 8, Cancer irroratus were exposed to three different doses of ethanol under an acute
paradigm or one exposure conducted daily for 5 days under a repeated paradigm. The alcohol
content of their hemolymph was determined using an enzymatic assay developed as part of this
work. Pericardial organ (PO) tissues were analyzed for changes in NP content using a
formaldehyde labeling scheme with 3-plex for acute samples and 2-plex for long-term samples.
NPs were quantified using a modified version of the DIA MS/MS technique developed in Ch. 6
that allowed for relative quantitation of the isotopic peaks, and every known small-medium-sized
crab NP was quantified using this method for long-term exposure samples. Significant changes
were observed for 77 of the 475 NPs, and 22 of those had changes greater than 25%. These NPs
were then quantified in short-term exposure PO extracts. After exposure to 0.2M ethanol, 12
were significantly increased compared to control. At 0.125M and 0.5M exposure, the values
341
were not significantly different from control. This profile, with an intermediate dose showing the
highest levels of NPs, suggests two factors working in opposition. Although it is unclear whether
or not alcohol is a rewarding stimulus for crabs, it is hypothesized that ethanol reduces NP
release by inhibition of basal levels of secretion due to its activation of the GABA receptor. It is
also causing an increase in the amount of NPs released at high doses, so another pathway may be
stimulating secretion. However, these effects may also be an artifact of too few experimental
animals in some conditions.
9.2 Future Directions
This work provides the foundation for many possible projects. The affinity-enhanced MD
could be applied in many types of experiments where NP concentration is limited, and similarly
there are many potential applications for the DIA MS/MS quantitation methods developed in this
dissertation. Of great significance is that all of the DIA MS/MS data could be re-analyzed to
quantify any peptide of interest, should additional ones become interesting.
The Orconectes rusticus neuropeptidome is expected to be useful in a study of the
neuroendocrine mechanisms by which this invasive species adapts rapidly to new environmental
conditions and out-competes native crayfish. A project quantifying NP changes in crayfish
collected from different lakes with different environmental conditions is currently underway.
Additional experiments are being conducted comparing different rearing conditions and genetic
factors on behavior in O. rusticus. These animals could also be subjected to neuropeptidomics
analysis. Behavioral experiments assessing response to predators and social dominance are
commonly conducted in this species, and NPs could be quantified from crayfish after predator
exposure or changes in social hierarchy [1-4]. An additional application could be conditioning of
crayfish to express a place preference for drugs of abuse, as has been conducted by other
342
researchers [5-9], followed by analysis of NP changes in this potentially new animal model of
addiction behavior.
Further refinement of feeding experiment parameters, including a quantitative assessment
of patterns in this behavior, would be of great benefit, along with additional replicates and a
shorter time scale of analysis. The animal could be exposed to different components of the
feeding experience to dissect the effects of NPs on each. For instance, it could be given a nonfood item to manipulate with its claws, exposed to the smell of food, or given an infusion of
glucose into the hemolymph to determine separately the effects of motor, chemical sensation,
and energy level changes that occur during feeding. Characterization of circadian changes in NPs
in the crab and under other experimental conditions would be of great interest as well. A NP
could be infused to determine what other NPs it causes the release of in vivo, and what
behavioral effects it has. Potentially feeding-related peptides could be administered to crabs that
are full but have access to food to determine the potency of their effects. Behavioral analysis of
the rewarding or aversive effects of ethanol in crabs would be of great use, along with NP
analysis of other neural organs. NP microdialysis could be conducted on crabs during ethanol
exposure or exposure to another environmental toxin they might be more likely to encounter. A
NP with large changes in one of the studies could be infused into the animal via MD concurrent
with monitoring behavior and collecting samples to analyze NP release in order to obtain more
information about its function. The applications of these techniques are far-reaching. Finally,
these techniques could be translated into mammalian models of disease for incorporation into a
traditional behavioral neuroscience workflow.
The research presented in this dissertation has advanced capabilities to study NPs in a
variety of species and under a myriad of physiological or behavioral manipulations. It has also
343
advanced our capabilities in MS/MS analysis by permitting simultaneous identification and
quantification from a single instrument run in a data set that can be mined over and over to
determine concentration changes in NPs. Opportunities also exist to implement other analytical
tools in this system for analysis of different types of compounds. The work conducted in this
dissertation has the potential to have great impact on the methodology used for behavioral
neuroscience.
9.3. Works Cited
1. Acquistapace, P.; Hazlett, B. A.; Gherardi, F. Unsuccessful predation and learning of predator cues by crayfish. J
Crustacean Biol. 2003, 23, 364-370.
2. Klocker, C. A.; Strayer, D. L. Interactions among an invasive crayfish (Orconectes rusticus), a native crayfish
(Orconectes limosus), and native bivalves (Sphaeriidae and Unionidae). Northeast Nat. 2004, 11, 167-178.
3. Martin, A. L.; Moore, P. A. Field observations of agonism in the crayfish, Orconectes rusticus: shelter use in a
natural environment. Ethology. 2007, 113, 1192-1201.
4. Martin, A. L.; Moore, P. A. The influence of dominance on shelter preference and eviction rates in the crayfish,
Orconectes rusticus. Ethology. 2008, 114, 351-360.
5. Dziopa, L.; Imeh-Nathaniel, A.; Baier, D.; Kiel, M.; Sameera, S.; Brager, A.; Beatriz, V.; Nathaniel, T. I.
Morphine-conditioned cue alters c-Fos protein expression in the brain of crayfish. Brain Research Bulletin.
2011, 85, 385-395.
6. Huber, R. Amines and motivated behaviors: a simpler systems approach to complex behavioral phenomena.
Journal of Comparative Physiology a-Neuroethology Sensory Neural and Behavioral Physiology. 2005,
191, 231-239.
7. Nathaniel, T. I.; Huber, R.; Panksepp, J. Repeated cocaine treatments induce distinct locomotor effects in
Crayfish. Brain Research Bulletin. 2012, 87, 328-333.
8. Nathaniel, T. I.; Panksepp, J.; Huber, R. Drug-seeking behavior in an invertebrate system: Evidence of morphineinduced reward, extinction and reinstatement in crayfish. Behavioural Brain Research. 2009, 197, 331-338.
9. Panksepp, J. B.; Huber, R. Ethological analyses of crayfish behavior: a new invertebrate system for measuring the
rewarding properties of psychostimulants. Behavioural Brain Research. 2004, 153, 171-180.
344
Appendix A. Protocols
A.1. Marine Crustacean Protocols
A.1.1. Acquisition and Housing
Jonah crabs (Cancer borealis) were obtained from the following three vendors: The Fresh
Lobster Company (Gloucester, MA), James Hook and Co. (Boston, MA), and Ocean Resources
(Sedgwick, ME). Male Jonah crabs were typically obtained, with the exception of a few
shipments from Ocean Resources. Male and female rock crabs (Cancer irroratus) were obtained
from Ocean Resources. Jonah and rock crabs were primarily caught by commercial fishermen
either as a primary catch in crab pots or as bycatch in lobster pots. Some rock crabs were caught
by hand by a diver. Female blue crabs (Callinectes sapidus) were obtained from local markets
(Asian Midway Foods, Madison, WI; Garden Asian Market, Middleton, WI) or The Crab Place
(Crisfield, MD). Animals obtained from distant vendors were shipped on ice packs with either
wet newspaper or seaweed via overnight air mail. Animals from local vendors arrived at the store
in boxes of wet newspaper. We took care to select the liveliest of the animals available, and put
them in tanks as soon as possible.
Animals were maintained in one of two aquarium systems, described in detail in the
thesis of Joshua Schmidt [1]. One system was comprised of a commercial tank, complete with
pump and filter. The other system employed a modified sump filtration system with protein
skimmer. All tanks had crushed gravel as substrate and water quality was monitored regularly.
Regular water changes were conducted and animals were fed if they were in the tanks for more
than 1 week. Animals were fed white fish, shrimp, crayfish, or crab meat, and were allowed to
eat for several hours. The room had temperature, humidity, and light controls. The tanks were
maintained at a water temperature of 10-15°C, and salinity was maintained in the 32 to 38 ppt
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range. The room was on a 12-hr reverse light schedule, with lights off from 10 AM to 10 PM—in
order to observe animals during their active period (dark) during normal work hours—and on
from 10 PM to 10 AM.
A.1.2. Hemolymph Withdrawal
Hemolymph was obtained from some animals via the following method, adapted from the thesis
of J. Schmidt [1] and other publications [2, 3]. Several items of practical relevance based on
personal experience have been noted here.
A.1.2.1. Materials
Crab (applicable species: Cancer borealis, Cancer irroratus, Callinectes sapidus and Carcinus
maenas), needle (25 or 26 gauge, ½” long, Beckton Dickinson and Co. (BD), Franklin Lakes,
NJ), plastic 1 mL or 3 mL syringe (BD), sample tubes, ice, aluminum dissecting pan
(ThermoFisher Scientific, Waltham, MA), crab saline (440 mM NaCl, 26 mM MgCl2, 13 mM
CaCl2, 11.2 mM Trizma base, 11 mM KCl, 5.1 mM maleic acid, pH adjusted to 7.4)
A.1.2.2. Instructions
1. Prepare the needle and syringe. It was determined that the smaller gauge needles, despite a
slight tendency to clog when proteins precipitate from the hemolymph, were preferable due to
producing less damage to the animal. Needles of ½ inch in length were also preferred to 1 inch or
1 ½ inch needles due to the tendency of larger needles to occasionally cause internal damage to
vital organs.
2. Remove the animal from the tank and either place with ventral side up in a metal dissecting
pan or hold with the thumb and forefinger of the experimenter’s non-dominant hand on the tail
section. This hold will make most species unable to use their chelae to harm the experimenter. C.
sapidus can have a longer reach and may be able to reach this area, and it should also be noted
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that the pereiopods of the crab are able to reach and potentially dig into the experimenter’s hand.
On C. irroratus, they are particularly sharp. Crabs can be anesthetized on ice for 15 min
depending on the needs of the experiment.
3. Uncap the needle and insert at one of the soft joints. The optimal location for a hemolymph
withdrawal varies depending on species, amount of hemolymph needed, and experimenter
preference. Typically, the best location is between the legs where they join the body. This allows
access to the central compartment of the animal, which contains more hemolymph that is in
direct contact with neuroendocrine and other organs. The first joint of the legs can also be
sampled, and for C. sapidus, the joint where the cheliped meets the body can be accessed from
the dorsal side of the animal. It is thus possible to place C. sapidus in the pan with dorsal side up,
tucking the legs underneath the body (and unable to harm the experimenter), and obtain
hemolymph from this joint. Regardless of placement, place the needle into the soft tissue and
withdraw the plunger on the syringe to create a vacuum. Change the orientation of the needle
inside the body until hemolymph is observed to enter the syringe. Once a good location is
obtained, the syringe and needle are held still until either the desired amount of hemolymph is
collected or the flow of hemolymph into the syringe ceases, even with additional pressure
applied via the plunger. Multiple locations may need to be used to obtain enough hemolymph.
4. Replace the volume of hemolymph taken if necessary. Using a new needle (and syringe if
desired), draw up a volume of crab saline equal to the volume of hemolymph taken. Inject this
into a soft joint near the central compartment of the animal. Removing 0.5 mL of hemolymph or
more each day will result in the animal’s death after 3-4 days, so if this volume or more is taken,
it is recommended to inject an equal volume of crab saline into the animal to replace lost volume.
Large volumes of hemolymph withdrawn may result in the animal’s death even if the volume is
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replaced. The estimated total hemolymph volume of the larger crabs (Cancer spp.) is 7 mL. C.
sapidus has a hemolymph volume closer to 5 mL, and C. maenas has around 3 mL. The
hemolymph of C. sapidus has a thicker consistency and a tendency to clot more rapidly than the
other species and should be stabilized soon after by precipitation of proteins with acidified
methanol (see A.3.2.2.).
A.1.3. Crab Dissection
Modified from the thesis of J. Schmidt [1]. A brief description follows but the primary document
should be consulted for more detailed instructions.
A.1.3.1. Materials
Crab (applicable species: Cancer borealis, Cancer irroratus, Callinectes sapidus, and Carcinus
maenas), ice, bucket, aluminum dissecting pan (ThermoFisher Scientific, Waltham, MA), bone
cutters (Fine Science Tools (FST), Foster City, CA), semimicro spatula with one round end and
one tapered end (ThermoFisher), dissecting scissors: sharp, straight ended, fine or extra fine size
(FST), toothed tissue forceps or curved micro mosquito hemostat (FST), curved scissors (15-20
total length, FST), crab saline (see A.1.2.), glass dish filled with black sylgard (ThermoFisher),
straight pins (FST), fine dissecting forceps (Dumont #55 straight recommended, FST), spring
scissors (5 mm cutting edge recommended, FST), dissecting microscope and light source,
microtubes, acidified methanol solution (90/9/1 MeOH/H2O/acetic acid), pipettes.
A.1.3.2. Instructions
1. Place the crab on ice for 15 min to anesthetize.
2. Remove the eyestalks and place in sylgard-filled dissection dish with crab saline on ice.
3. Remove the top portion of the carapace and dissect out the stomach.
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4. Cut this open and rinse in crab saline prior to placing flat in the dissection dish with crab
saline on ice.
5. Remove the pericardial ridges and rinse in crab saline. Keep in crab saline on ice until
microdissection under a dissecting microscope.
6. Cut open the eyestalks and find the sinus gland (SG). It is immediately below the retina and is
a white disc-shaped tissue. Remove with scissors and forceps.
7. Pin the stomach flat in the dish (inside of stomach down) and locate the brain. It will be
underneath the epidermis near the rostral end. Remove by cutting the nerves going into this
tissue.
8. To dissect the pericardial organ, first cut the lateral part of the pericardial ridges so that they
can lay flat, with the side facing the heart up. Pin in place.
9. Locate the pericardial organ underneath a membrane that covers portions of the inside of the
pericardial ridges. It will be a filamentous white structure with a more bluish or iridescent tint
compared to muscle tissue.
10. Follow the length of the PO throughout the ridge by holding it gently with forceps in one
hand and cutting any connective tissue with the spring scissors in the other hand. A diagram of
the PO should be referred to in order to obtain as much of the organ as possible.
11. Put dissected tissues immediately into tubes with acidified methanol on ice.
A.2. Orconectes rusticus Protocols
A.2.1. Collection
Orconectes rusticus were kindly provided by Alex Laztka, a graduate student in the
laboratory of Jake Vander Zanden, in the Department of Limnology at the University of
Wisconsin-Madison, and his co-workers. They were obtained from fresh water lakes in Northern
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Wisconsin centered around the Limnology Department’s Trout Lake Research Station, on Trout
Lake in Boulder Junction, WI. Animals were sampled as part of a National Science Foundation
(NSF)-funded Long-Term Ecological Research (LTER) study, and were obtained and
transported with the permission of the Wisconsin Department of Natural Resources. Permission
for transport must be obtained due to the status of O. rusticus as an invasive species in
Wisconsin. Regulations on their transport have been in flux over the last several years so the
DNR’s website should be consulted. A recreational fishing permit is also recommended. The
trapping method has been shown to preferentially collect male crayfish, and although both
genders were collected in our study, around 75% of the animals were male.
A.2.1.1. Materials for Catching Crayfish
Gee minnow trap (Aquatic Sampling Company, Buffalo, NY), scissors, rope, buoy, bait (beef
liver or chicken liver from local butcher), bucket with lid, refrigerator, Styrofoam cooler, ice
packs, tape
A.2.1.2. Procedure for Catching Crayfish
1. Modify the minnow trap (see Fig. A.1.A.) by increasing the entrance hole to ~5cm to
accommodate mature male crayfish.
2. Bait the trap and assemble the two halves into a whole trap (See Fig. A.1.B.). Secure the two
halves with the clip.
3. Attach the clip to the buoy by 1m of rope.
4. Place the trap in the lake at a depth of approximately 1 m, in an area where the lake substrate
is “cobble,” or rocks approximately 6-8 cm in diameter.
5. In 20-24 hrs, remove the trap.
6. Dump crayfish from the trap into a bucket.
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7. Separate O. rusticus from other catch by hand and place into a Styrofoam cooler with ice
packs. Native crayfish, hybrid crayfish, and juvenile fish may also be caught. The fish and native
crayfish should be released. Rusty crayfish are easily distinguished from native species by the
presence of two large red spots on the carapace (see Fig. A.2.). It may also be useful to sort by
gender/form at this point. O. rusticus should not be released, even if non-desired genders/forms
are caught. There are two forms of males, the reproductive form I and the non-reproductive form
II. They can be distinguished by the appearance of their reproductive gonopods (See Fig. A.3.).
Form I males have feathery tips on the gonopods, whereas form II males have more solid
gonopods, often with orange-colored tips. Form I males also have hooks on some leg segments.
The female has gonoducts and lacks gonapods. Individuals not desired as samples should be
kept, euthanized, and disposed of. Coolers should be taped shut during transport to prevent
escape. Transporting to Madison, WI by personal vehicle (a trip of approximately 3.5 hrs) in this
manner did not lead to significant mortality.
8. Keep animals in coolers with ice packs or buckets with lids in a refrigerator at 4ºC for
approximately 12 hrs. The crayfish should not be stored in water, as the water will quickly
become foul and deoxygenated. Storing animals in water in the refrigerator and/or during
transport led to approximately 95% mortality in a batch of around 350 crayfish. Stored dry and
cold, they enter a state similar to hibernation and can survive longer.
A.2.2. Housing Crayfish
A.2.2.1. Materials for Housing Crayfish
Crayfish, food for crayfish (commercial bottom feeder pellets, fresh fish, etc.), freshwater
aquarium setup: 20 gal tank with close-fitting lid, water, crushed gravel, filter (Aquaclear 30,
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Rolf C. Hagen, Inc., Montreal, Canada), air pump, air line, air stone, fish net breeder isolation
box (Lee’s Aquarium and Pet Products, San Marcos, CA)
A.2.2.2. Housing Crayfish
Suitable aquaria will contain fresh—preferably reverse osmosis—water and crushed
gravel substrate. They also should be equipped with appropriate filtration and oxygenation
devices, in our case aquarium hobbyist supplies including a filtration unit rated for a tank 10 gal
larger than actually used, and an air bubbler with tubing and air stone. No more than 45
individuals should be housed per 20 gallon tank. Care should be taken to ensure a tight fit for the
lid of the aquarium as the crayfish can escape through very small holes. Animals should not be
stored more than a day at a density of 45 animals/20 gal tank, but lower densities, about 20 per
20 gal tank, can be housed indefinitely.
For long-term care of the animals, O. rusticus should be fed and monitored for molting,
and regular water changes should be conducted. Crayfish will cannibalize if they are not fed
every 2-3 days, and fighting often occurs over food. They can be fed raw meat of any type, but
white fish is recommended as it does not foul the water. Pelleted food designed for bottomfeeding fish can also be purchased from a pet store and fed to the crayfish. It must be the kind
that will sink to the bottom without falling apart, and usually comes in a disc shape. Enough food
should be presented so that all animals have something to eat to prevent fighting. If O. rusticus
molt, they should be isolated with the old shell in an isolation net box until their new shell
hardens. Other individuals will attack and kill unprotected newly molted individuals. These
newly molted individuals can and should consume the old shell for nutritional purposes.
Hardening takes 1-2 days. If males and females are housed together, mating may occur. If this is
undesirable, separate the genders. Waste will accumulate in the substrate and water, so it is
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advisable to clean the tank weekly with a 20% water change, or more often if housed in high
density.
Euthanasia of crayfish can be accomplished by freezing, putting on ice for an extended
period of time, or submerging in hot tap water. Bodies of animals should be frozen prior to
disposal to ensure that they are not carrying viable eggs. Boiling is an alternative method both
for euthanasia and destruction of embryos.
A.2.3. Orconectes rusticus Dissection
Brain and eyestalk can be dissected from O. rusticus without the aid of a microscope. It is
recommended to prepare whole eyestalk extracts instead of dissecting the sinus gland out of the
eyestalk due to its miniscule size, which is difficult to locate even under a microscope.
A.2.3.1. Materials
Crayfish, ice, aluminum dissecting pan (ThermoFisher Scientific, Waltham, MA), dissecting
scissors: sharp, straight ended, fine or extra fine size (Fine Science Tools (FST), Foster City,
CA), fine forceps (FST), crayfish saline (195 mM NaCl, 13.5 mM CaCl2, 11.2 mM Trizma base,
5.4 mM KCl, 5.1 mM maleic acid, 2.6 mM MgCl2, pH adjusted to 7.4), sample tubes, acidified
methanol (90/9/1 MeOH/H2O/acetic acid v/v/v).
A.2.3.2. Dissection
1. Put crayfish on ice until movement ceases (approximately 15-20 min).
2. Place animal ventral side up.
3. Use scissors to remove appendages and cut the tail nerve to prevent tail flipping (Fig. A.4.)
4. Place the animal dorsal side up.
5. Remove antennules and antennae. (Fig. A.5.A.)
6. Snap off the rostrum (Fig. A.5.B.
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7. Remove the eyestalks by cutting at their base, where the stalk meets the shell (Fig. A.6.). Set
aside in acidified methanol.
8. Cut the carapace along a line starting below where one eyestalk was located, continuing up
toward the rostrum, and then down toward the other eyestalk’s previous location (Fig. A.7.).
9. Gently pull the rostral portion away. (Fig. A.8.)
10. Locate the brain inside the shell, immediately caudal to the rostrum. (Fig. A.9.)
11. Remove with fine forceps and scissors. (Fig. A.10.) Place in a 1.5 mL tube containing 0.5 mL
acidified methanol (90:9:1 methanol: water: acetic acid by volume) on ice. 25-30 brains will fit
in a single tube of this size. About 20-25 pairs of eyestalks will fit in each tube.
12. Store tissues at -20°C or -80°C (if more than a month will elapse) until extraction.
A.3. Sample Preparation Protocols
A.3.1. Tissue Extraction
A.3.1.1. Materials for Tissue Extraction
Tissues, acidified methanol (90:9:1 MeOH:H2O:Acetic acid), microtubes, plastic pestles or glass
vial and pestle set, sonicator, microcentrifuge, speedvac, pipettes and tips
Note: Keep samples on ice whenever possible.
A.3.1.2. Tissue Extraction Procedure
1. For soft tissues, use a plastic pestle to grind the tissue in the collection tube into pieces as
small as possible. For whole eyestalks, place in a glass vial and grind with pestle until as
homogeneous as possible. Put the eyestalk homogenate back into a microtube. Vortex the
sample.
2. Sonicate the tube containing tissue and liquid for 5 min. Do not add heat.
3. Centrifuge 5 min x 16,100 g to separate liquid and solid portions.
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4. Remove supernatant and set aside in a new tube.
5. Add a volume of acidified methanol equal to the original liquid volume to the pellet.
6. Repeat steps 1-5 twice (for a total of 3 extractions) and combine the supernatants. The pelleted
solids can be saved, but it has been determined that the first and second extractions of most
tissues contain the most neuropeptides. Subsequent extractions contain primarily lipids and/or no
compounds of interest.
7. Evaporate the solvent using the speedvac. Medium or Low heat is recommended.
A.3.2. Hemolymph Extraction
A.3.2.1. Materials for Hemolymph Extraction.
Materials: Hemolymph, acidified methanol (90:9:1 MeOH:H2O:Acetic acid), solution for
resuspension, microtubes, plastic pestles (optional), sonicator, microcentrifuge, speedvac,
pipettes and tips
Note: Keep samples on ice whenever possible.
A.3.2.2. Hemolymph Extraction Steps
1. For alcohol studies, collect hemolymph and spin down (5 min x 16,100g). Freeze at -80°C if
necessary. Thaw and remove hemolymph for alcohol content analysis.
2. Take 0.5 mL of the remaining hemolymph and add an equal volume of acidified methanol.
Vortex vigorously. This can be stored at -20°C.
3. Sonicate the tube for 5 min. Do not use heat.
4. Centrifuge 5 min x 16,100 g to separate liquid and solid portions.
5. Remove supernatant and set aside in a new tube.
6. Add a 0.5 mL acidified methanol to the pellet and resuspend. Vortexing and/or a plastic pestle
can be used to aid in breaking up the pellet.
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7. Repeat steps 3-6 twice (for a total of 3 extractions) and combine the supernatants. The pelleted
solids can be saved, but it has been determined that the first and second extractions of most
tissues contain the most neuropeptides. Subsequent extractions contain primarily lipids and/or no
compounds of interest.
8. Evaporate the solvent using the speedvac. Medium or Low heat is recommended.
9. Resuspend the sample prior to storage at -20°C. The solvent and volume to use for
resuspending will be determined by the next sample preparation step. If MWCO is to be
conducted, 70/30 H2O/MeOH is used. If formaldehyde labeling is next, use H2O. If desalting is
next, use H2O with 0.1% FA. It is important to resuspend prior to freezing, because it has been
found that hemolymph samples frozen before resuspension will not achieve full dissolution.
A.3.3. Molecular Weight Cutoff (MWCO)
Hemolymph samples were usually subjected to molecular weight cutoff (MWCO)
following a combination of the manufacturer’s recommended procedure and a published
modification [4]. This step is conducted on the crude extracts prior to desalting or other steps. If
MWCO was not conducted, the hemolymph samples were typically too thick for efficient
desalting.
A.3.3.1. MWCO Materials
Amicon Utracel 10 kDa MWCO 0.5mL size, 70/30 H2O/MeOH, 50/50 H2O/MeOH,
0.1M NaOH, tubes, centrifuge, speedvac, pipette and tips
A.3.3.2. MWCO Procedure
1. Rinse MWCO device with 0.2mL 0.1M NaOH: add solution to tube and spin 4 min x 14,000g.
2. Rinse MWCO with 0.5 mL of 50/50 H2O/MeOH: spin 8 min x 14,000g.
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3. Load sample, dissolved in 0.5mL 30/70 MeOH/H2O (sonicate if necessary to resuspend), into
device, and run through the membrane. This will take 15-20 min x 14,000g.
4. Rinse sample tube with 0.1 mL of 30/70 MeOH/H2O, and run through the membrane. This
will take 5 min x 14,000g. This rinse is combined with the flow-through in step 3.
5. Both the flow-through and concentrate were saved, but only the flow-through was used for
neuropeptide analysis.
6. Evaporate the solvent for the flow-through using a speedvac (Low/Med heat).
A.3.4. Desalting
Some tissue and hemolymph extracts were subjected to a desalting step, especially prior
to analysis via MALDI ionization or formaldehyde labeling.
A.3.4.1. Desalting Materials
C18 Zip Tip pipette tips, 10 µL size (Millipore), acetonitrile, 0.1% FA in H2O, 50/50/0.1
ACN/H2O/FA, tubes, speedvac, pipettes and tips.
A.3.4.2. Desalting procedure
1. Wet the C18 bed in the tips with 15 µL of acetonitrile (ACN). (Draw up and expel to waste)
2. Equilibrate the tips with 15 µL of 0.1% FA in water. (Draw up and expel to waste)
3. Resuspend the sample in a minimal volume of 0.1% FA in water, up to 60 µL. Sonicate 5 min.
This can be done prior to wetting and equilibration of the tips.
4. Bind sample to the tip by drawing it up and expelling it back into the original tube 12 times,
with 15 µL moved through the C18 bed each time.
5. Rinse the C18 bed with 15 µL of 0.1% FA in water 4 times to remove salts and highly
hydrophilic compounds. For some samples, the first two rinses can be added back to the original
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sample in case re-analysis will be required and/or the sample is very precious. Other rinses were
expelled to waste.
6. Elute hydrophobic compounds, including peptides, from the C18 material by pulling up and
dispensing a solution of 50/50/0.1 ACN/H2O/FA in 15 µL amounts 6 times. For MALDI
analysis, a smaller volume can be used and the sample spotted directly onto the plate.
Alternatively, MALDI matrix solution in 50/50/0.1 ACN/H2O/FA can be used to elute the
peptides and spot immediately.
7. For UPLC analysis, dilute with up to 15 µL of 0.1% FA in water to prevent evaporation loss,
and add internal standard if necessary. For formaldehyde labeling, dry down using a speedvac.
A.3.5. Alternative Desalting Method Using C18 Magnetic Beads
For some samples, C18 magnetic beads were used for desalting instead of Zip Tips.
These had the advantage of rapid and easy separation from the liquid by holding a magnet to the
side of the tube for 30 s. This was particularly useful if samples were viscous and would not go
through Zip Tips easily.
A.3.5.1. Magnetic C18 Desalting Materials
Materials: C18 magnetic beads (Bioclone, Inc., San Diego, CA), magnet, 50/50 MeOH/H2O,
acetonitrile, 0.1% FA in H2O, 50/50/0.1 ACN/H2O/FA, tubes, speedvac, pipettes and tips.
A.3.5.2. Magnetic C18 Desalting Procedure
1. Suspend C18 beads in 50% MeOH/H2O at a final concentration of 2.5 g/mL. Store at 4°C.
2. Obtain a volume of beads equal to half of the sample volume.
3. Rinse beads 3 times with 2 times the bead volume of 0.1% FA in H2O. Beads are separated
from solution by holding a magnet to the side of the tube for 30 s, and the liquid is removed
without disturbing the particles. Beads are mixed with liquid by gentle vortexing.
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4. Add back the original bead volume of 0.1% FA in H2O.
5. Add the beads to the resuspended samples. Allow samples to bind for 2 min at room
temperature.
6. Remove the liquid portion and set aside (in case it needs to be re-analyzed)
7. Rinse the beads 3 times with a volume of 0.1% FA in H2O equal to that of the original sample.
The first rinse may sometimes be added back to the original liquid portion in the event that reanalysis is required, and/or the sample is very precious.
8. Elute peptides from the beads by incubating with a volume equal to ¼ of the original sample
volume of 50/50/0.1 MeOH/H2O/FA for 2 min at room temperature.
9. Save the liquid and repeat step 8 two times.
10. Prepare for LC analysis by adding 0.1% FA in H2O to prevent evaporation and internal
standard, or dry in the speedvac.
A.3.6. Isotopic Formaldehyde Labeling
Both two-plex [5] and a three-plex [6] formaldehyde labeling schemes were used. These
were modified from references. The two-plex uses formaldehyde (OCH3) and deuterium
formaldehyde (OCD3) as dimethylation agents, and the three-plex adds carbon-13 deuterium
formaldehyde (O13CD3) as an additional dimethylation agent. The two-plex protocol does not
add sodium ions to the sample due to the use of a milder H- donor, borane pyridine as opposed to
NaBH3CN. However, for the heaviest label, D- must be donated instead of H- to produce a total
mass shift of four, because simply using carbon-13 deuterium formaldehyde will only produce a
mass difference of two, which could have interference from naturally occurring isotopes of the
compound labeled with deuterium formaldehyde. For this reason, it uses NaBH3CN for the light
and intermediate label, and NaBD3CN for the heavy label. A light label incorporates 2(CH 2) per
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primary amine; an intermediate label incorporates 2(CD2) per primary amine; and a heavy label
incorporates 2(CD2-H+D) per primary amine. All peptide N-termini (except those blocked by
pyroglutamation or acetylation) and lysine sidechains will be labeled.
A.3.6.1. Two-Plex Formaldehyde Labeling Materials
Formaldehyde, 1% solution in water (v/v) (Sigma), D-formaldehyde, 1% solution in
water (v/v) (Isotech), borane pyridine, solution in isopropanol (Sigma), water, desalted sample of
neuropeptides from 1-3 organs, 100 mM ammonium bicarbonate solution, water bath at 37°C,
speedvac, pipette
A.3.6.2. Two-Plex Formaldehyde Labeling Instructions
1. Dissolve sample in ~10 µL of water. Sonicate to ensure full dissolution.
2. Prepare formaldehyde, deuterium formaldehyde, and borane pyridine solutions in a chemical
fume hood.
3. Add 10 µL of formaldehyde solution to samples for light labeling or 10µL of deuterium
formaldehyde solutions to samples for intermediate labeling.
4. Add 10 µL of borane pyridine solution to each sample.
5. Incubate at 37°C for 15 min in the water bath.
6. Take samples out of the water bath and add 10 µL 100 mM ammonium bicarbonate solution.
7. Dry samples in speedvac. Samples will be fully dry when they do not smell of ammonia. A
sample may look dry, but still contain ammonia. Highly basic conditions can cause peptide
breakdown, so it is necessary to remove all ammonia.
A.3.6.3 Materials for Three-Plex Formaldehyde Labeling
Formaldehyde, 1% solution in water (v/v) (Sigma), D-formaldehyde, 1% solution in
water (v/v) (Isotech), NaBH3CN solution in water (), NaBD3CN solution in water (), water,
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desalted sample of neuropeptides from 1-3 organs, 1% NH4OH solution in water (Fisher),
speedvac, pipette
A.3.7. Affinity Agents: Preparation, Elution of Bound Analytes, and In Vitro Bead-binding
Experiments
A.3.7.1. C18 Silica Microparticles
Materials: silica particles coated with octadecyl carbon (C18H37), Microsorb, 3 µM size, 300 Å
pore size, (Varian, now part of Agilent), 0.5% bovine serum albumin (BSA) in water solution
(w/v) with FA 0.1% (v/v), Handee Spin Cups with paper filter (Pierce, Thermo Fisher Scientific,
Rockford, IL), microcentrifuge, speedvac, 0.1% FA in H2O (v/v), tubes, pipettes and tips, vials
for UPLC autosampler (Waters, Millford, MA, and Micoliter Analytical Supplies, Suwanee, GA)
1. Dissolve C18 silica microparticles (C18SµP) in water with 0.5% BSA to aid solubility and
0.1% FA to improve NP binding to the beads.
2. Separate C18 silica beads from the solution by employing paper filter spin cups that fit into
1.5mL tubes. The solution containing the solid particles is loaded into the cup, which is inserted
into a tube. The tube is then centrifuged, upon which the liquid flows through the filter into the
tube, and the solid particles are retained.
3. Elute analyte from the C18SµP by incubating with a solution of 50/50/0.1 ACN/water/formic
acid (FA) and centrifuging.
4. Save the flow-through and add to the liquid recovered from washing the beads in order to
collect all analyte present in the dialysate. Dry down, resuspend in 0.1% FA, and place in UPLC
vials for further analysis.
A.3.7.2. Magnetic Beads
A.3.7.2.1. Materials for all Magnetic Beads
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Bio-Mag C18 magnetic beads (Bioclone Inc., San Diego, CA), protein G magnetic microbeads
(2 µM diameter, New England Biolabs, Ipswich, MA), protein G magnetic nanobeads (100 nm
diameter, Chemicell, Berlin, Germany), neodymium magnet, rabbit polyclonal anti-FMRFamide
antibody (Abcam, Cambridge, MA), phosphate buffered saline (PBS, 140 mM NaCl, 8mM
Na2HPO4, 2mM KH2PO4, 10mM KCl)., vortexer, oscillating platform, peptide standards
(American Peptide, Sunnyvale, CA), 0.1% FA in H2O (v/v), 2% FA in H2O (v/v), 50/50/0.1
ACN/H2O/FA (v/v/v), tubes, pipettes and tips, 96-well plate, 1 mL round, with lid mat (Waters,
Millford, MA)
Magnetic particles were separated from solution as described previously by holding a
neodymium magnet to the side of the tube for 30 s prior to removing the liquid by pipette.
Protocols for their preparation and use were developed starting with the manufacturers’
instructions.
A.3.7.2.2. C18 Magnetic Beads
1. Prepare as in protocol A.3.5.
2. For in vitro binding assessments, incubate 100 µL beads with 100 µL peptide solution at
room temperature for 2 min.
3. Rinse 3 times with 300 µL of PBS or 0.1% FA. These liquid portions may be saved for
determination of which analytes did not bind to the particles.
4. Elute analytes from beads by incubating for 2 min at room temperature with 100 µL of
50/50/0.1 ACN/H2O/FA. This may be analyzed directly by UPLC-MS if desired.
A.3.7.2.3. Antibody-linked Magnetic Microparticles
1. Rinse 100 µL magnetic beads in solution as provided by the manufacturer three times with
300 µL of PBS.
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2. Add the antibody at a final dilution of 1:500 (ratio of 50µL beads: 1µL of a 1:10 dilution of
serum). In this instance, 2 µL of a 1:10 dilution of antibody serum would be added to the beads
along with 98 µL of PBS.
3. Incubate with gentle shaking (attached to vortexer at lowest setting) at 4º C overnight.
4. Rinse 3 times with 300 µL of PBS.
5. Store in 100 µL of PBS.
6. For in vitro binding efficacy assessment, incubate beads with 100 µL of the peptide standard
solution (standards in the 10-5-10-6M range) at room temperature with gentle agitation
(oscillating platform) for 30 min.
7. Rinse 3 times with 300 µL of PBS to remove unbound analyte. These rinses may be saved and
analyzed for peptide content to determine what does not bind to the antibody-coated beads.
8. To elute bound analyte, incubate with 100 µL of 2% FA in H2O at room temperature for 10
min. This may be analyzed directly by UPLC-MS if desired.
A.3.7.2.4. Antibody-coated Magnetic Nanoparticles
1. Rinse 100 µL magnetic beads in solution as provided by the manufacturer three times with
300 µL of PBS.
2. Add the antibody at a final dilution of 1:500 (ratio of 50µL beads: 1µL of a 1:10 dilution of
serum). In this instance, 2 µL of a 1:10 dilution of antibody serum would be added to the beads
along with 98 µL of PBS. An alternate, higher dilution of 1:50 can be used, in this case 10 µL of
a 1:10 dilution of serum and 90 µL of PBS added to the beads.
3. Incubate with gentle shaking (attached to vortexer at lowest setting) at room temperature for
15 min.
4. Rinse 3 times with 300 µL of PBS.
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5. Store in 100 µL of PBS.
6. For in vitro binding efficacy assessment, incubate beads with 100 µL of the peptide standard
solution (standards in the 10-5-10-6M range) at room temperature with gentle agitation
(oscillating platform) for 15 min.
7. Rinse 3 times with 300 µL of PBS to remove unbound analyte. These rinses may be saved and
analyzed for peptide content to determine what does not bind to the antibody-coated beads.
8. To elute bound analyte, incubate with 100 µL of 2% FA in H2O at room temperature for 2
min. This may be analyzed directly by UPLC-MS if desired.
Note that nanoparticles will aggregate if they are sonicated, placed on ice, or allowed to dry out.
A.4. Myoglobin Digest Internal Standard
Equine skeletal myoglobin (ERA, Colden, CO) was digested with trypsin (bovine pancreas,
Sigma-Aldrich, St. Louis, MO) following a published procedure [7]. Myoglobin was dissolved in
100 mM NH4HCO3 at 0.5 mg/mL. Trypsin was dissolved in the same solution at 1 µg/mL and
added to the myoglobin solution at an enzyme: substrate ratio of 1:10. This was then diluted 1:1
with methanol and placed in a water bath at 37°C for 45 min. The reaction was stopped by
adding ice-cold acetic acid to a final concentration of 5%. The sample was then spun at 15,100 x
g for 5 min.
A.5. Microdialysis
A.5.1. Materials and Preparation
CMA/20 Elite probes with 4 mm membranes of polyarylether sulfone (PAES) (CMA
Microdialysis (Harvard Apparatus, Holliston, MA, USA)); syringe pump: CMA/102, KD
Scientific 100 (KD Scientific Inc., Holliston, MA, USA), Harvard22 (Harvard Apparatus,
Holliston, MA, USA); FEP tubing (CMA), PEEK tubing (Upchurch-Scientific, Idex Health and
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Science, Oak Harbor, WA, USA); flanged connectors: CMA, BASi (West Lafayette, IN, USA);
syringe: BD (Franklin Lakes, NJ, USA) plastic 1 mL or 3 mL; 21 gauge Luer-lock needles
(included with CMA 20 series probes) with the sharp points removed by a grinding wheel on a
rotary tool (Dremel, Robert Bosch LLC, Farmington Hills, MI, USA); water; crab saline; peptide
standards (American Peptide, Sunnyvale, CA); tubes; pipettes and tips
1. Rinse MD probe with water or crab saline prior to use and store in liquid throughout the study
to maintain the integrity of the membrane.
A.5.2. In Vitro Microdialysis
Additional materials: tubes, 1000µL pipette tip, scissors, orbital rotating platform, phosphate
buffered saline (PBS), 96-well plate, 1 mL round, with lid mat (Waters, Millford, MA)
1. Immerse the tip of the probe or probes into a vial via a home-built apparatus to hold the probes
in place. For a single probe, a 1000µL pipette tip can be cut in half and the top part placed inside
a 1.5 mL tube. The probe is placed inside this holder.
2. Put microdialysis medium in the vial. This is a solution of phosphate buffered saline (PBS)
solution with neuropeptide standards of interest in it at known concentrations in the micromolar
range.
3. The entire vial with probe holder is placed on an orbital rotating platform to produce constant
mixing of the microdialysis medium.
4. Obtain a sample of the medium prior to starting microdialysis and set aside.
5. Start flow of the perfusing liquid (0.5 µL/min) via the syringe pump. Permit the probe to
equilibrate at the flow rate of the experiment for 30 min before starting dialysate collection.
6. Collect technical replicates as consecutive 30 min samples of the liquid flowing out of the
tubing. A minimum of three should be collected.
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7. Obtain a sample of the medium after all technical replicates are collected. Samples from the
medium taken before and after the experiment were used to determine the relative recovery
percentage.
8. Medium samples and dialysate can be placed immediately in a 96-well sample plate for
UPLC-QTOF analysis.
A.5.3. In Vitro Microdialysis-Affinity enhanced
Additional materials: affinity agent (see protocol above), ball bearing (1/8 inch, Wheels
Manufacturing, Louisville, CO, USA), rocking platform
1. Set up apparatus as in steps 1-3, with the exception that the probe is immersed in PBS alone.
2. Put a clean ball bearing into the syringe.
3. Prepare perfusate with affinity agent by diluting 50µL of bead solution (as it arrived from the
manufacturer) to 3mL (a dilution of 1:60) or to 0.5mL (a 1:10 dilution) with PBS and put into
syringe. Although the concentrations of beads in mg/mL vary depending on the affinity agent, it
was determined that they have equal activity per mL, as the all manufacturers’ protocols
recommend the same ratio of beads to sample, i.e. 50µL beads with 0.5 mL cell lysate.
4. Put syringe in pump and place on rocking platform at an angle to the axis of rotation. Start the
platform to rock, causing the ball bearing to roll up and down the syringe.
5. Start perfusing the system at a higher flow rate until 3 times the volume of the system has
passed through the tubing.
6. Place the probe in microdialysis medium as in step 2 above. Obtain a sample of the medium
and set aside.
7. Start the pump at the flow rate desired (0.5 µL/min). Allow equilibration as in step 5 of
previous protocol. Collect samples as in steps 6 and 7. Put medium samples in plate as in step 8.
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8. For samples containing affinity agent, separate solids from perfusate liquid, which can be
placed in the 96-well plate. Elute bound NPs from the solid particles according to each particle’s
instructions and add to the 96-well plate.
A.5.4. Crab Microdialysis
A.5.4.1. Crab Microdialysis (MD) Surgery
Additional materials: crab, dissection tray, ice, 30% bleach, ethanol, rotary tool (Dremel, as
above), 1/32” drill bit for Dremel, hot glue and hot glue gun, food coloring, Loctite super glue
gel (Loctite, Westlake, OH), Mighty Putty (epoxy materials suspended in a clay-like base,
Mighty Putty, North Wales, PA), plastic electrical cord protector (from hardware store),
refrigerated fraction collector (BASi Honeycomb, Bioanalytical Systems, Inc. Indianapolis, IN).
Figures are provided: See Figs. A.11-19. Place a crab on ice for 15 min to anesthetize. Depth of
anesthesia is monitored by watching for voluntary movement. It is maintained by keeping the
crab on ice throughout the surgery.
2. Wash the crab’s shell in the area immediately above the pericardial sinus with 30% bleach
followed by ethanol. Scrubbing should be vigorous and conducted until color no longer appears
to come off. This can be done while the crab is being anesthetized.
3. Score the shell in this area by making cross-hatching diagonal lines using a grinding wheel on
a rotary tool.
4. Apply a small amount of hot glue to the MD probe on the shaft above the membrane area to
mark the deepest point to which the probe will be inserted. The tubing for the probe should
already be connected and the integrity of the tubing and probe can be checked throughout
surgery by gently depressing the syringe plunger. Artificial crab saline should be the perfusate.
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5. Drill a 1/32” hole in a location estimated to be immediately above the heart. Do not apply
pressure to the drill bit while drilling, and stop once the shell has been drilled through. If
hemolymph does not come out of the hole, the epidermis has not been punctured. Use the needle
that comes with the probe to gently puncture the epidermis. Dry off the shell
6. Carefully insert the probe. This should stop hemolymph from leaking out of the hole.
7. Apply Loctite super glue gel in a small circle around the probe.
8. Mix the Mighty Putty and apply on top of this glue circle. It is best to mix the material in the
last few minutes of anesthesia induction, and form a log of approximately 2” in length with the
diameter of a pen. This can then be placed on the shell and molded into a cone shape around the
exposed part of the probe.
9. Fill any remaining voids with super glue gel.
10. Insert the probe tubing into a 1’ section of plastic tubing for protecting electrical cords.
11. Attach this tubing to the putty cone on the crab’s shell with hot glue.
12. Keep the crab on ice until the putty is no longer pliable, and then replace in the tank. The
surgery should not take more than 45 min, and a typical surgery is around 25 min. The crab may
not wake up if it is kept on ice for longer than 45 min.
13. Start the syringe pump and refrigerated fraction collector as desired.
14. When starting an experiment, determine the dead volume from the probe tip to collection vial
as follows and incorporate this information to collect the proper samples.
Tubing volume: 1.8 µL/10cm, probe tip to outlet: 3.0 µL, fraction collector needle: 7.1 µL
15. After the experiment is concluded, which be no more than 10 days after surgery, sacrifice the
crab. The crab can be kept for other experiments if desired but the probe will probably not work
anymore.
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16. Follow the normal dissection protocol with the following changes.
17. While the crab is on ice, cut the MD tubing and protective tubing short and connect a syringe
with 0.1mL of diluted food coloring (dilute 1:1 v:v) to the inlet tube.
18. Manually push the dye through the tubing and through the outlet tube. Make sure at least a
few drops go all the way through.
19. When dissecting, be careful not to disturb the probe inside the shell.
20. At the point when you remove the shell over the pericardial region, take additional care. Note
the location of the probe and/or dye spot. There should be a spot of dye on the crab’s heart.
A.5.4.2. Crab Microdialysis-Affinity Enhanced
1. Set up syringe as indicated for in vitro affinity-enhanced MD, with the exception that crab
saline is used to dilute the affinity agent instead of PBS.
2. Perfuse the system with three times its dead volume, as above, prior to equilibration and
sample collection.
3. Collect samples manually into tubes suspended over ice. It is not recommended to use the
fraction collector for AE-MD due to the potential for clogging and interactions between the
magnetic beads and metal fraction collector needle. The tubes should not be immersed in ice, as
this causes nanoparticles to aggregate.
A.6. Instrumental Analysis
A.6.1. Packing Chemically-Fritted C18 Columns with Integrated Tips
Materials: Waters nanoAcquity UPLC system (Millford, MA), polyimide-coated glass capillary,
360 µM OD, 75 µm ID (Polymicro Technologies, Phoenix, AZ), Magic C18 particles (Michrom,
Auburn, CA, USA), 3 µm diameter, 100Å pore size), potassium silicate (Kasil®, PQ
Corporation, Malvern, PA) , formamide (Sigma-Aldrich), tetramethylammonium silicate (15-
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20% in water, Sigma-Aldrich), column packing setup and gas cylinder (manufactured by UW
Chemistry Shop, similar products available commercially), 2 mL vial or LC sample vial,
ultramicro stir bar, sonicator, various other fittings (IDEX Health & Science LLC), capillary
cutter, P-2000 Laser Puller (Sutter Instruments, Novato, CA), methanol, isopropanol, soldering
iron, 0.1% FA in ACN (Optima or LC-MS grade), 0.1% FA in H2O (Optima or LC-MS grade)
1. Cut a section of capillary ~15 cm long. Burn off ~5 cm of polyimide coating from the middle,
creating a clear window. Note: capillaries are much more fragile after the protective polyimide
coating has been removed.
2. Mix together 100 µL potassium silicate and 100 µL tetramethylammonium silicate.
3. Add 20 µL formamide.
4. Heat up the soldering iron.
5. Dip the end of the capillary into the silicate solution. Capillary action will draw the solution
up, Allow it to reach past the window.
6. Touch the tip of a hot soldering iron to the places where you want to make frits. This should
be within the clear window, but closer to the part that is coated. The laser must shine on the
middle to properly pull the tip and you don’t want anything else to be in that area. The area
touched should briefly turn white within 5 sec. Once you see it turn white, take the soldering iron
off. If it doesn’t turn white, you probably didn’t see it happen. Holding the soldering iron there
too long can cause the frit to be too solid.
7. Rinse the capillary with methanol using the bomb or acetonitrile using the UPLC system.
8. Dry the capillary with the bomb or a syringe fitted with the proper adapter.
Note: It is frequently useful to make several at one time, as the frit solution must be used within a
few hours of being made, and these things are very fragile so it is helpful to have spares. Do not
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allow the capillaries to sit for more than an hour with the frit solution in them, as it will start to
set up.
9. Prepare column packing material. This should be ~10mg of C18 particles in 1mL of 3:1
methanol: isopropanol. Add the stir bar and sonicate for >15 sec.
10. Put the packing material in the vial in the bomb and close the bomb.
11. Put the capillary to be packed into the bomb. Make sure the end of the capillary is only a few
mm above the stir bar by pushing it down until it hits the bottom of the vial, and then taking it
back out a few mm.
12. Tighten the capillary in the bomb and close the vent valve.
13. Put pressure on the regulator and ~100psi on the bomb. You should start to see small clumps
of C18 particles travelling up the capillary and stopping at the frit, and liquid should come out
the top.
14. Once it is packed ~2cm, put full pressure on the bomb ( up to 1500 psi). Allow the column to
pack for ~40 min. You can’t see part of the column because it is inside the bomb, so you will
have to let it pack for longer than you expect.
15. Start to vent the bomb slowly. First, turn the pressure off at the regulator. Then open the vent
valve very slowly. You should only barely be able to hear gas escaping. Sometimes you can
check by putting an unstretched glove over the outlet—if it balloons up, gas is flowing out.
Venting should take at least 3 hrs.
16. Attach the column to the UPLC outlet.
17. Rinse the column with 95%B at a flow rate that gives you a pressure of ~3000psi.
18. Reduce the flow rate and switch to 50/50, then increase again to obtain 3000psi.
19. Do the same thing for 95%A. Allow it to pack at 95%A and 3000 psi for at least 15 min.
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20. Return the column to 50/50 and unhook it.
21. Dry the column on the air bomb.
22. Pull the tip. The other side will make a nice ESI emitter tip for the Synapt.
A.6.2. Analytical Scale UPLC for Pre-Fractionation
Materials: neutral pH solvents (7.4, solvent A: 25mM NH4HCO2, solvent B: 9:1 ACN: 25mM
NH4HCO2), reversed-phase Inertsil ODS-4 column, 3µm, 2.1 x 150 mm (GL Sciences, Tokyo,
Japan), Alliance HPLC with UV detection (Waters, Milford, MA), Rainin Dynamax Model FC-4
fraction collector, tubes, speedvac
1. Inject sample on a 120 min RP separation that will run 95%A to 5%A and back.
2. Collect fractions every 3 min after the first 5 min have elapsed.
3. Dry samples down with speedvac.
4. Combine early eluting fractions with late eluting fractions to reduce the total number of
samples while maintaining separation.
A.6.3. UPLC-MS and UPLC-MS/MS Analysis
Materials: Waters nanoAcquity UPLC system, Zorbax 300SB-C18, 300 µm x 5 mm trap column
(Agilent Technologies Inc., Santa Clara, CA), holder for 5 mm trap cartridges (Agilent), stainless
steel nano-tee (IDEX Health & Science LLC, Oak Harbor, WA), conductive filter capsule with 2
µm filter and holding bracket (IDEX), QTOF Micro (Micromass, Cambridge, UK), Synapt G2
(Waters), home packed column, C18 reverse-phase column (BEH130 C18, 1.7 µm, 75 µm x 100
mm, Waters), trap column (Waters), home-pulled ESI tips, 0.1% FA in ACN (Optima or LC-MS
grade), 0.1% FA in H2O (Optima or LC-MS grade)
1) Set up UPLC conditions. Typically, an acidic reversed phase gradient with 0.1% FA in water
and 0.1% in ACN as mobile phase was employed with run time ranging from 25 min to 120 min
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depending on the application. The change in gradient ranged from 5%B to 95%B or 10%B to
45%B (for highly hydrophilic peptides). A trapping step is employed, ranging in length from 1
min for clean samples to 5 min for very salty samples. For the Synapt, a commercial column is
used with a column heater and a separate home-pulled capillary emitter tip. For the QTOF micro,
a home-packed column with integrated emitter tip is used.
2) Set up MS conditions.
Micromass (currently Waters) QTOF micro: can be run in MS only mode over the mass range
m/z 500-700 for highly accurate quantitation of a few known peptides in that mass range. DDA
can also be run on this instrument but it is not recommended due to sensitivity issues. This
instrument is recommended for high concentration samples and/or standards.
Waters Synapt G2 mass spectrometer: For DIA MS/MS mode, use a high energy scan having a
voltage ramp from 25 to 65 V. For DDA, use a basic file that selects 3 precursors at a time. Use
Glu-fibrinopeptide for lockspray calibration.
A.7. Data Analysis
A.7.1. QTOF Micro Data Analysis
1. Use QuanLynx software (Waters). Input the m/z’s of interest for your analytes manually.
2. Once the QuanLynx method is created, use it to analyze your data. The data must be in the
QuanLynx project folder, you must be using the QuanLynx project, and you must have the
correct file names in the sample list.
3. Manually check the identity of each peak. To do this, open a chromatogram in MassLynx and
generate the XIC for the m/z you are looking at. Combine spectra across the peak and search for
the analyte in the spectrum. Ensure it is high intensity, not an isotope of another compound, and
has the correct charge state.
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4. Manually check the integration of each peak.
5. Export results to Excel and or JMP Statistical Software (Version 9.0.2 SAS Institute, Inc.,
Cary, NC, USA).
A.7.2. Synapt G2 Data Analysis—DDA
1) Load results into ProteinLynx Global Server (Waters). Make sure it is configured to output a
.pkl file (one per sample) and note the location where these are created.
2) Create a preprocessing file to correct with the lockspray.
3) Attach the preprocessing file to the raw data and run. This will generate the .pkl file.
4) This file can be uploaded to Mascot or PEAKS for database search and/or de novo
sequencing. The following PTMs are used: C-terminal amidation, pyroglutamic acid, methionine
oxidation. No enzyme. Sometimes Cys dehydration is also used.
5) Export results to Microsoft Excel.
A.7.3. Synapt G2 Data Analysis—DIA
1) If desired, generate a .pkl using the correct preprocessing file. This can be searched in Mascot
but cannot be used in PEAKS.
2) More typically, quantitation will be conducted. Generate the appropriate quantitation file in
Skyline SRM software [8]
(https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view).
The website has downloads and helpful tutorial videos and documents. I have created the
following files: 475 crab NPs labeled with H and D formaldehyde, 475 crab NPs unlabeled,
475+CPRPs and other long NPs unlabeled, NPs from Ruibing’s feeding paper [5] and myoglobin
tryptic digest, sets of neuropeptide standards, NPs from long-term ethanol exposure labeled with
3-plex dimethylation.
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3) Upload your data files into Skyline.
4) Manually check integrations for alignment with predicted retention time, retention time
alignment across replicates, choice of the correct peak, uniqueness of the peak for that peptide,
etc. The retention time comparison and peak area comparison windows can be helpful for this.
5) Export desired results. I have made an export file that I like to use called CMSexport. Analyze
in Excel and/or JMP. Make sure to sort peptides by name and sum all transitions from the same
peptide.
Additional Skyline settings: Modifications were added to peptides manually. A non-specific
enzyme type was used, with cleavage at every amino acid, and 9 missed cleavages permitted, the
most allowed. Skyline does not have a “no enzyme” option built in and this is the closest
approximation possible. Most NPs are shorter than 9 amino acids, this should be acceptable. In
addition, Skyline will quantify peptides that do not meet its filter criteria if they are input
manually and the exception to the filters is noted by the user. The iRT function with retention
time predictor was used, calibrated off of results from Mascot identifications of (Matrix Science,
London, UK) equine myoglobin tryptic peptides in a previous LC run of the myoglobin digest
alone under the same conditions. The 2+ and 3+ precursors were monitored to the 4 most
abundant 1+ and 2+ b and y ions, and in some cases (CCAP, somatostatin-14) the precursor ion
itself was also monitored. MS/MS filtering parameters were set to DIA with MSe isolation, and
10,000 resolving power.
A.8. Alcohol Dehydrogenase (ADH) Assay (with thanks to Lauren Putterman)
A.8.1. Materials
Alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, ≥300 units/mg protein (Sigma
Aldrich, St. Louis, MO), phenazine methosulfate (PMS, Sigma Aldrich), β-nicotinamide adenine
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dinucleotide hydrate (NAD+), thiazolyl blue tetrazolium bromide (MTT), ethanol 100% (Sigma
Aldrich), crab, 1mL plastic syringe with ½” 26 ga needle (Becton Dickinson, Franklin Lakes,
NJ), crab saline, centrifuge, tubes, ice, laboratory incubator/oven capable of 50°C, microplate
reader capable of reading absorbance at 570 and 655nm: Tecan Ultra 384 with XFluor4 software
(Tecan Group Ltd., Männedorf, Switzerland) with filters from Omega Optical (Omega Optical,
Inv., Brattleboro, VT), microplates (96-well Perkin Elmer Isoplate, white frame and clear well,
Perkin Elmer, Waltham, MA), phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1
mM NaH2PO4, 1.76 mM KH2PO4, pH 7.4), tris buffer (0.1 M Tris-HCl, 0.1% (w/v) Triton X100, pH 8.5), sodium dodecyl sulfate (SDS) 20% (w/v) in 50/50 dimethylformamide (DMF)/H2O
(v/v), rotating platform (Fisher Scientific Clinical Rotator), pipettes (multi-channel are useful)
Note: Several kinds of ADH are available. Some have NADH in them, and are not suitable for
this assay. Sigma Adrich product # A3263 is suitable.
Storage notes:
Plasma from unaffected crabs can be stored for weeks in the -80ºC freezer. Calibration curves
are also stable for up to 2 weeks. Tris buffer should be stored at 4°C. MTT should be stored at 6
mM concentration in PBS in the dark at room temperature. This solution can be kept for up to a
month. For each assay, dilute with PBS to 600 µM; do not keep this solution more than a day.
ADH (7.33x106 U/L) and NAD+ (37.7 mM) solutions should be made up weekly and stored at
4°C. PMS is stored at 50 mM or 50 µM in at 4°C in the dark. These solutions are stable for up to
a month. A solution of 5 pM PMS is made on the day of the experiment and not stored.
SDS/DMF should not be stored in the refrigerator and is stable for several months.
A.8.2. Assay Procedure
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1. Collect hemolymph from the crab as described previously. A very small amount is required
for this assay, but additional hemolymph from an un-dosed crab will be needed to make blanks
and calibration curve samples. Hemolymph should be put on ice immediately after it is collected.
2. Spin down at 16.1 rcf for 5 minutes to get crab plasma, keep on ice. The plasma can be stored,
especially “blank” plasma. For “blank” plasma, it appears to be stable for several weeks.
3. Turn on oven (50 °C).
4. Make calibration curve solutions in crab plasma. It is easier to make a 100 mM EtOH solution
first, then dilute to 10 mM. Use this to make 5, 3, and 1 mM solutions in plasma. Use the 1 mM
solution to make a 0.1 mM solution.
5. To each well of the plate to be used, add 76.7 uL of Tris-HCl Buffer
6. Add 10 uL of 600 uM MTT to each well.
7. Add 10 uL of 37.7 mM NAD+ solution to each well.
8. Add 4 uL of sample to each well. This should include a calibration curve of 0.1 mM, 1 mM, 3
mM, 5 mM, and 10 mM, sometimes done in duplicate, and a blank in addition to the actual
samples. Samples from crabs exposed to alcohol should also be diluted 1:3 with Tris buffer and
analyzed at this concentration to ensure they fit within the assay’s linear range.
9. Add 2 uL of 5 pM PMS to each well.
10. Add 4 uL of 7.33x106 U/L active ADH to each well
11. Mix plate on rotating platform for about 1 minute.
12. Place plate in 50 °C oven/incubator for 60 minutes.
13. Stop the reaction by adding 100 uL of inhibitor (SDS/DMF solution) to each well.
14. Mix plate on rotating platform for about 1 minute.
15. Determine the absorbance at 570 nm and 655 nm using the plate reader.
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16. The software will output a .csv file that can be analyzed in Microsoft Excel.
A.8.3. ADH Assay Analysis
1. Adjust for the blank, if not already done during data acquisition.
2. Determine the difference between absorbance at 570 nm and 655 nm.
3. Plot the concentration of ethanol versus the 570 nm-660 nm absorbance value and generate a
linear fit. Sometimes, the 10 mM point will have to be excluded from the fit.
4. Determine the slope and intercept of the linear regression. Use these values to calculate the
concentrations of the unknown samples (concentration = (absorbance – intercept)/slope). If the
values fall outside of the range for which the calibration curve was generated, the values cannot
be trusted. Dilute samples if necessary.
A.9. Works Cited
1. Schmidt, J. J. From crabs to hamsters: Bioanalytical mass spectrometry for peptidomic analysis and biomarker
discovery [3279009]. United States -- Wisconsin: The University of Wisconsin - Madison; 2007.
2. Behrens, H. L.; Chen, R.; Li, L. Combining microdialysis, NanoLC-MS, and MALDI-TOF/TOF to detect
neuropeptides secreted in the crab, Cancer borealis. Anal Chem. 2008, 80, 6949-6958.
3. Chen, R.; Ma, M.; Hui, L.; Zhang, J.; Li, L. Measurement of neuropeptides in crustacean hemolymph via MALDI
mass spectrometry. J Am Soc Mass Spectrom. 2009, 20, 708-718.
4. Cunningham, R.; Wang, J.; Wellner, D.; Li, L. Investigation and reduction of sub-microgram peptide loss using
molecular weight cut-off fractionation prior to mass spectrometric analysis. J Mass Spectrom. 2012, 47,
1327-1332.
5. Chen, R.; Hui, L.; Cape, S. S.; Wang, J.; Li, L. Comparative Neuropeptidomic Analysis of Food Intake via a
Multi-faceted Mass Spectrometric Approach. ACS Chem Neurosci. 2010, 1, 204-214.
6. Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J. R. Multiplex peptide stable isotope
dimethyl labeling for quantitative proteomics. Nat Protoc. 2009, 4, 484-494.
7. Li, F.; Schmerberg, C. M.; Ji, Q. C. Accelerated tryptic digestion of proteins in plasma for absolute quantitation
using a protein internal standard by liquid chromatography/tandem mass spectrometry. Rapid Commun
Mass Spectrom. 2009, 23, 729-732.
8. MacLean, B.; Tomazela, D. M.; Shulman, N.; Chambers, M.; Finney, G. L.; Frewen, B.; Kern, R.; Tabb, D. L.;
Liebler, D. C.; MacCoss, M. J. Skyline: an open source document editor for creating and analyzing targeted
proteomics experiments. Bioinformatics. 2010, 26, 966-968.
A.10. Figures
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A.1. Modified minnow trap for collection of Orconectes rusticus
A.
B.
A. Half of a trap, showing the enlarged hole for entry of crayfish, several crayfish in the trap, and
the remains of the bait after a day in the water (pinkish blob). B. A full assembled trap. Hooks
and loops interlock on opposite halves, and two loops line up for securing with a pin-like
fastener. That fastener is attached to a small buoy (whitish oval) by a length of string. This trap
has been pulled out of the lake after a day, and contains several crayfish along with small fish
bycatch.
Figure A.2. Identification of Orconectes rusticus
A.
B.
Rusty crayfish are primarily green or brown, with red or orange accents. Dark reddish spots are a
defining feature on the carapace. Arrows indicate the location of these spots in A. overhead and
B. side views.
379
Figure A. 3. Genders and Stages of Orconectes rusticus
b.
c.
b.
a.
a.
d.
Male form I, form II, and female adult O. rusticus (Left to right). Males have gonapods (a.), but
those of the form I male are more feathery at the ends (b.). Male form II gonapods are more
substantial and often orange at the ends. Male form I crayfish also have hooks on their legs (c.).
Females lack gonapods and instead have a gonoduct (d.).
Figure A.4. Dissection Step 3
A.
A. Severing the tail nerve. B. Removing the legs.
B.
380
Figure A.5. Dissection Step 5-6
A.
B.
A. Removing anntennules and antennae. C. Snapping off the rostrum.
Figure A.6. Dissection Step 7
A
.
B
.
A. Cut the eyestalk at the base. B. A better view of the exposed eyestalks.
Figure A.7. Dissection Step 8.
381
Make a cut all the way around the carapace as indicated.
Figure A.8. Dissection Step 9
Separate the rostral portion of the shell.
Figure A.9. Dissection Step 10
Locate the brain inside the rostral portion of the shell. It is the whitish structure the arrow is
pointing to.
Figure A.10.
A.
B
.
382
A. Cut the nerves surrounding the brain and holding it in place. B. Remove with forceps.
Figures A.11-19. Microdialysis
383
384
385
386
387
Appendix B. Crustacean Neuropeptide Database
>gi|001 |LiLab|LL001
AAPYAFGL
>gi|002 |LiLab|LL002
AASPYSFGL
>gi|003 |LiLab|LL003
AGGAYSFGL
>gi|004 |LiLab|LL004
AGLYSYGL
>gi|005 |LiLab|LL005
ARPYAFGL
>gi|006 |LiLab|LL006
DGPYSFGL
>gi|007 |LiLab|LL007
DPYAFGLRHTSFVLYAFGL
>gi|008 |LiLab|LL008
DRPYSFGL
>gi|009 |LiLab|LL009
EPYEFGL
>gi|010 |LiLab|LL010
ERPYSFGL
>gi|011 |LiLab|LL011
FNGCNFGL
>gi|012 |LiLab|LL012
FSGASPYGL
>gi|013 |LiLab|LL013
FSGTYNFGL
>gi|014 |LiLab|LL014
GDPYAFGL
>gi|015 |LiLab|LL015
GGAYSFGL
>gi|016 |LiLab|LL016
GKPYAFGL
>gi|017 |LiLab|LL017
GPYSFGL
>gi|018 |LiLab|LL018
GQYAFGL
>gi|019 |LiLab|LL019
GRYSFGL
>gi|020 |LiLab|LL020
GSGQYAFGL
>gi|021 |LiLab|LL021
HGTEGPYPFGL
>gi|022 |LiLab|LL022
HSPSSASYDFGL
>gi|023 |LiLab|LL023
KLPYSFGL
>gi|024 |LiLab|LL024
LKAYDFGL
>gi|025 |LiLab|LL025
LVKYSFGL
>gi|026 |LiLab|LL026
NPYSFGL
>gi|027 |LiLab|LL027
Allatostatin A-type
AAPYAFGLamide Cmaen Br/PO
Allatostatin A-type
AASPYSFGLamide Cmaen PO
Allatostatin A-type
AGGAYSFGLamide Cb br,PO
Allatostatin A-type
AGLYSYGLamide Cb PO
Allatostatin A-type
ARPYAFGLamide Cmaen PO
Allatostatin A-type
DGPYSFGLamide Csap PO,br Cb PO
Allatostatin A-type
DPYAFGLRHTSFVLYAFGLamide Csap
Allatostatin A-type
DRPYSFGLamide Cb PO
Allatostatin A-type
EPYEFGLamide Cmaen Br
Allatostatin A-type
ERPYSFGLamide Cp.PO/Cb.PO.
Allatostatin A-type
FNGCNFGLamide Csap
Allatostatin A-type
FSGASPYGLamide Cmaen Br/PO
Allatostatin A-type
FSGTYNFGLamide Csap PO,br
Allatostatin A-type
GDPYAFGLamide Cb br,PO
Allatostatin A-type
GGAYSFGLamide Cb.PO.
Allatostatin A-type
GKPYAFGLamide Cmaen Br/PO
Allatostatin A-type
GPYSFGLamide Cb PO
Allatostatin A-type
GQYAFGLamide Cb.PO.
Allatostatin A-type
GRYSFGLamide Csap br
Allatostatin A-type
GSGQYAFGLamide Cb.PO.
Allatostatin A-type
HGTEGPYPFGLamide Csap
Allatostatin A-type
HSPSSASYDFGLamide Cb PO
Allatostatin A-type
KLPYSFGLamide Cmaen Br
Allatostatin A-type
LKAYDFGLamide Cmaen Br
Allatostatin A-type
LVKYSFGLamide Cb PO,CoG
Allatostatin A-type
NPYSFGLamide Csap PO,br Cb PO
Allatostatin A-type
PADLYEFGLamide Cb.PO.
388
PADLYEFGL
>gi|028 |LiLab|LL028 Allatostatin A-type PATDLYAFGLamide Cb.PO.
PATDLYAFGL
>gi|029 |LiLab|LL029 Allatostatin A-type PDMYGFGLamide Cb.PO.
PDMYGFGL
>gi|030 |LiLab|LL030 Allatostatin A-type
pQRAYSFGLamide Csap PO Cb
,br,PO,CoG
QRAYSFGL
>gi|031 |LiLab|LL031 Allatostatin A-type pQRDYSFGLamide Cb PO
QRDYSFGL
>gi|032 |LiLab|LL032 Allatostatin A-type pQRPYSFGLamide Cp.PO
QRPYSFGL
>gi|033 |LiLab|LL033 Allatostatin A-type pQRTYSFGLamide Cb.PO.
QRTYSFGL
>gi|034 |LiLab|LL034 Allatostatin A-type
PRDYAFGLamide Csap PO,br Cb
br,PO,STG,CoG
PRDYAFGL
>gi|035 |LiLab|LL035 Allatostatin A-type PRVYSFGLamide Csap PO
PRVYSFGL
>gi|036 |LiLab|LL036 Allatostatin A-type PSMYAFGLamide Cb.PO.
PSMYAFGL
>gi|037 |LiLab|LL037 Allatostatin A-type RGPYAFGLamide Cmaen Br/PO
RGPYAFGL
>gi|038 |LiLab|LL038 Allatostatin A-type SGHYIFGLamide Csap PO
SGHYIFGL
>gi|039 |LiLab|LL039 Allatostatin A-type SKSPYSFGLamide Cb br,PO
SKSPYSFGL
>gi|040 |LiLab|LL040 Allatostatin A-type SNPYSFGLamide Csap PO
SNPYSFGL
>gi|041 |LiLab|LL041 Allatostatin A-type SPRLTYFGLamide Csap PO
SPRLTYFGL
>gi|042 |LiLab|LL042 Allatostatin A-type SSGQYAFGLamide Csap PO Cb PO
SSGQYAFGL
>gi|043 |LiLab|LL043 Allatostatin A-type SYAFGLamide Cb PO
SYAFGL
>gi|044 |LiLab|LL044 Allatostatin A-type TAPYAFGLamide Csap PO
TAPYAFGL
>gi|045 |LiLab|LL045 Allatostatin A-type TPHTYSFGLamide Csap
TPHTYSFGL
>gi|046 |LiLab|LL046 Allatostatin A-type
TRPYSFGLamide Csap PO Cmaen
Br/PO
TRPYSFGL
>gi|047 |LiLab|LL047 Allatostatin A-type VGPYAFGLamide
VGPYAFGL
>gi|048 |LiLab|LL048 Allatostatin A-type YAHSFGLamide Csap br
YAHSFGL
>gi|049 |LiLab|LL049 Allatostatin A-type AST-3 GGSLYSFGLamide
GGSLYSFGL
>gi|050 |LiLab|LL050 Allatostatin A-type Carcinustatin 1, Penaeustatin 13
YAFGLamide Cb.PO.
YAFGL
>gi|051 |LiLab|LL051 Allatostatin A-type Carcinustatin 10 APQPYAFGLamide
Cmaen PO Cb br
APQPYAFGL
389
>gi|052 |LiLab|LL052 Allatostatin A-type Carcinustatin 11 ATGQYAFGLamide
Cmaen Br
ATGQYAFGL
>gi|053 |LiLab|LL053 Allatostatin A-type Carcinustatin 12 PDMYAFGLamide
Cb.PO.
PDMYAFGL
>gi|054
|LiLab|LL054
Allatostatin
A-type
Carcinustatin
13
EYDDMYTEKRPKVYAFGLamide Cmaen PO
EYDDMYTEKRPKVYAFGL
>gi|055 |LiLab|LL055 Allatostatin A-type Carcinustatin 14, Penaeustatin
35 YSFGLamide Cp.PO/Cb.PO.
YSFGL
>gi|056 |LiLab|LL056 Allatostatin A-type Carcinustatin 15, Procastatin 27
AGPYSFGLamide Csap PO,br Cmaen Br/PO Cb PO
AGPYSFGL
>gi|057 |LiLab|LL057 Allatostatin A-type Carcinustatin 16 GGPYSYGLamide
GGPYSYGL
>gi|058 |LiLab|LL058 Allatostatin A-type Carcinustatin 17, Procastatin 10
SGQYSFGLamide
SGQYSFGL
>gi|059 |LiLab|LL059 Allatostatin A-type Carcinustatin 18 SDMYSFGLamide
Cmaen PO Cb PO
SDMYSFGL
>gi|060 |LiLab|LL060 Allatostatin A-type Carcinustatin 19 APTDMYSFGLamide
APTDMYSFGL
>gi|061 |LiLab|LL061 Allatostatin A-type Carcinustatin 2 EAYAFGLamide
Cmaen Br/PO
EAYAFGL
>gi|062
|LiLab|LL062
Allatostatin
A-type
Carcinustatin
20
GYEDEDEDRPFYALGLGKRPRTYSFGLamide Cmaen PO
GYEDEDEDRPFYALGLGKRPRTYSFGL
>gi|063 |LiLab|LL063 Allatostatin A-type Carcinustatin 3 EPYAFGLamide
Cmaen Br/PO Cb br,PO
EPYAFGL
>gi|064 |LiLab|LL064 Allatostatin A-type Carcinustatin 4 DPYAFGLamide
Cmaen Br Cb br,PO
DPYAFGL
>gi|065 |LiLab|LL065 Allatostatin A-type Carcinustatin 5 NPYAFGLamide
Cmaen Br/PO
NPYAFGL
>gi|066 |LiLab|LL066 Allatostatin A-type Carcinustatin 6 SPYAFGLamide
SPYAFGL
>gi|067 |LiLab|LL067 Allatostatin A-type Carcinustatin 7 ASPYAFGLamide
Cmaen Br/PO
ASPYAFGL
>gi|068 |LiLab|LL068 Allatostatin A-type Carcinustatin 8, Penaeustatin 6,
Procastatin 17 AGPYAFGLamide Csap br Cmaen Br/PO
AGPYAFGL
>gi|069 |LiLab|LL069 Allatostatin A-type Carcinustatin 9 GGPYAFGLamide
Cmaen Br
GGPYAFGL
>gi|070 |LiLab|LL070 Allatostatin A-type Lepidopteran peptide cydiastatin
1 SPHYNFGLamide
SPHYNFGL
390
>gi|071 |LiLab|LL071 Allatostatin A-type Lepidopteran peptide cydiastatin
2, helicostatin 2 [11-18] LPVYNFGLamide
LPVYNFGL
>gi|072 |LiLab|LL072 Allatostatin A-type Lepidopteran peptide cydiastatin
2, helicostatin 2 AYSYVSEYKRLPVYNFGLamide
AYSYVSEYKRLPVYNFGL
>gi|073 |LiLab|LL073 Allatostatin A-type Lepidopteran peptide cydiastatin
3,helicostatin 3 SRPYSFGLamide Cb PO
SRPYSFGL
>gi|074 |LiLab|LL074 Allatostatin A-type Lepidopteran peptide cydiastatin
4, helicostatin 4 ARPYSFGLamide Csap PO Cmaen Br/PO Cb PO
ARPYSFGL
>gi|075 |LiLab|LL075 Allatostatin A-type Lepidopteran peptide cydiastatin
5 ARGYDFGLamide Csap PO
ARGYDFGL
>gi|076 |LiLab|LL076 Allatostatin A-type Lepidopteran peptide cydiastatin
6 LPLYNFGLamide
LPLYNFGL
>gi|077 |LiLab|LL077 Allatostatin A-type Lepidopteran peptide cydiastatin
7 KMYDFGLamide
KMYDFGL
>gi|078
|LiLab|LL078
Allatostatin
A-type
Lepidopteran
peptide
helicostatin 1 SPHYDFGLamide
SPHYDFGL
>gi|079
|LiLab|LL079
Allatostatin
A-type
Lepidopteran
peptide
helicostatin 5 ARAYDFGLamide Csap PO
ARAYDFGL
>gi|080
|LiLab|LL080
Allatostatin
A-type
Lepidopteran
peptide
helicostatin 6 LPMYNFGLamide Cb.PO.
LPMYNFGL
>gi|081
|LiLab|LL081
Allatostatin
A-type
Lepidopteran
peptide
helicostatin 7 ARSYNFGLamide
ARSYNFGL
>gi|082
|LiLab|LL082
Allatostatin
A-type
Lepidopteran
peptide
helicostatin 8 YSKFNFGLamide
YSKFNFGL
>gi|083
|LiLab|LL083
Allatostatin
A-type
Lepidopteran
peptide
helicostatin 9 ERDMHRFSFGLamide
ERDMHRFSFGL
>gi|084
|LiLab|LL084
Allatostatin
A-type
Lepidopteran
peptide
Lepidostatin I AKSYNFGLamide
AKSYNFGL
>gi|085
|LiLab|LL085
Allatostatin
A-type
Penaeustatin
1
ANEDEDAASLFAFGLamide
ANEDEDAASLFAFGL
>gi|086 |LiLab|LL086 Allatostatin A-type Penaeustatin 10 TPSYAFGLamide
TPSYAFGL
>gi|087 |LiLab|LL087 Allatostatin A-type Penaeustatin 11 pQRDYAFGLamide
QRDYAFGL
>gi|088 |LiLab|LL088 Allatostatin A-type Penaeustatin 12 SDYAFGLamide
SDYAFGL
>gi|089 |LiLab|LL089 Allatostatin A-type Penaeustatin 14 ANQYTFGLamide
ANQYTFGL
>gi|090 |LiLab|LL090 Allatostatin A-type Penaeustatin 15 ASQYTFGLamide
391
ASQYTFGL
>gi|091 |LiLab|LL091 Allatostatin A-type Penaeustatin 16 SQYTFGLamide
SQYTFGL
>gi|092 |LiLab|LL092 Allatostatin A-type Penaeustatin 17 YTFGLamide
YTFGL
>gi|093 |LiLab|LL093 Allatostatin A-type Penaeustatin 18 SGHYNFGLamide
Csap PO
SGHYNFGL
>gi|094 |LiLab|LL094 Allatostatin A-type Penaeustatin 19 GHYNFGLamide
GHYNFGL
>gi|095
|LiLab|LL095
Allatostatin
A-type
Penaeustatin
2
PDAEESNKRDRLYAFGLamide
PDAEESNKRDRLYAFGL
>gi|096 |LiLab|LL096 Allatostatin A-type Penaeustatin 20 AGPYEFGLamide
AGPYEFGL
>gi|097 |LiLab|LL097 Allatostatin A-type Penaeustatin 21 GGPYEFGLamide
GGPYEFGL
>gi|098 |LiLab|LL098 Allatostatin A-type Penaeustatin 22 AAPYEFGLamide
Cmaen Br/PO
AAPYEFGL
>gi|099 |LiLab|LL099 Allatostatin A-type Penaeustatin 23 GPYEFGLamide
GPYEFGL
>gi|100 |LiLab|LL100 Allatostatin A-type Penaeustatin 24 SPYEFGLamide
SPYEFGL
>gi|101 |LiLab|LL101 Allatostatin A-type Penaeustatin 25 NPYEFGLamide
NPYEFGL
>gi|102
|LiLab|LL102
Allatostatin
A-type
Penaeustatin
26
NEVPDPETERNSYDFGLamide
NEVPDPETERNSYDFGL
>gi|103
|LiLab|LL103
Allatostatin
A-type
Penaeustatin
27
EVPDPETERNSYDFGLamide
EVPDPETERNSYDFGL
>gi|104
|LiLab|LL104
Allatostatin
A-type
Penaeustatin
28
PETERNSYDFGLamide
PETERNSYDFGL
>gi|105 |LiLab|LL105 Allatostatin A-type Penaeustatin 29 NSYDFGLamide
NSYDFGL
>gi|106 |LiLab|LL106 Allatostatin A-type Penaeustatin 3 DRLYAFGLamide
DRLYAFGL
>gi|107 |LiLab|LL107 Allatostatin A-type Penaeustatin 30 YDFGLamide
YDFGL
>gi|108 |LiLab|LL108 Allatostatin A-type Penaeustatin 31 AGHYSFGLamide
AGHYSFGL
>gi|109 |LiLab|LL109 Allatostatin A-type Penaeustatin 32 DRTYSFGLamide
DRTYSFGL
>gi|110 |LiLab|LL110 Allatostatin A-type Penaeustatin 33 PSAYSFGLamide
PSAYSFGL
>gi|111 |LiLab|LL111 Allatostatin A-type Penaeustatin 34 pQNMYSFGLamide
QNMYSFGL
>gi|112
|LiLab|LL112
Allatostatin
A-type
Penaeustatin
36
DARGALDLDQSPAYASDLGKRIGSAYSFGLamide
DARGALDLDQSPAYASDLGKRIGSAYSFGL
>gi|113
|LiLab|LL113
Allatostatin
A-type
Penaeustatin
37
TARGALDLDQSPAYASDLGKRIGSAYSFGLamide
392
TARGALDLDQSPAYASDLGKRIGSAYSFGL
>gi|114 |LiLab|LL114 Allatostatin A-type
SVAYGFGL
>gi|115 |LiLab|LL115 Allatostatin A-type
TVAYGFGL
>gi|116 |LiLab|LL116 Allatostatin A-type
TGGPYAFGL
>gi|117 |LiLab|LL117 Allatostatin A-type
GIYGFGL
>gi|118 |LiLab|LL118 Allatostatin A-type
SAGPYAFGL
>gi|119 |LiLab|LL119 Allatostatin A-type
SGHYAFGL
>gi|120 |LiLab|LL120 Allatostatin A-type
ANQYAFGL
>gi|121 |LiLab|LL121 Allatostatin A-type
AGQYAFGL
>gi|122 |LiLab|LL122 Allatostatin A-type
QNNYGFGL
>gi|123 |LiLab|LL123 Allatostatin A-type
PRNYAFGL
>gi|124
|LiLab|LL124
Allatostatin
TSDEEDDEDDQYYPYGLamide
TSDEEDDEDDQYYPYGL
>gi|125 |LiLab|LL125 Allatostatin A-type
PRVYGFGL
>gi|126
|LiLab|LL126
Allatostatin
ADSYGLAFGNGGDALEMGLamide
ADSYGLAFGNGGDALEMGL
>gi|127 |LiLab|LL127 Allatostatin A-type
SYDFGL
>gi|128 |LiLab|LL128 Allatostatin A-type
TAGPYAFGL
>gi|129 |LiLab|LL129 Allatostatin A-type
SGPYAFGL
>gi|130 |LiLab|LL130 Allatostatin A-type
TGPYAFGL
>gi|131 |LiLab|LL131 Allatostatin A-type
TPNYAFGL
>gi|132 |LiLab|LL132 Allatostatin A-type
ADPYAFGL
>gi|133 |LiLab|LL133 Allatostatin A-type
PNPYAFGL
>gi|134 |LiLab|LL134 Allatostatin A-type
DGMYSFGL
>gi|135 |LiLab|LL135 Allatostatin A-type
AGQYSFGL
>gi|136 |LiLab|LL136 Allatostatin A-type
SGPYSFGL
>gi|137 |LiLab|LL137 Allatostatin A-type
EDYDSSDQYSL
>gi|138 |LiLab|LL138 Allatostatin A-type
SGAYSFGL
>gi|139 |LiLab|LL139 Allatostatin A-type
Penaeustatin 38 SVAYGFGLamide
Penaeustatin 39 TVAYGFGLamide
Penaeustatin 4 TGGPYAFGLamide
Penaeustatin 40 (X)GIYGFGLamide
Penaeustatin 5 SAGPYAFGLamide
Penaeustatin 7 SGHYAFGLamide
Penaeustatin 8 ANQYAFGLamide
Penaeustatin 9 AGQYAFGLamide
Procastatin 1 QNNYGFGLamide
Procastatin 11 PRNYAFGLamide
A-type
Procastatin
12
Procastatin 13 PRVYGFGLamide
A-type
Procastatin
14
Procastatin 15 SYDFGLamide
Procastatin 16 TAGPYAFGLamide
Procastatin 18 SGPYAFGLamide
Procastatin 19 TGPYAFGLamide
Procastatin 2 TPNYAFGLamide
Procastatin 20 ADPYAFGLamide
Procastatin 21 PNPYAFGLamide
Procastatin 22 DGMYSFGLamide
Procastatin 23 AGQYSFGLamide
Procastatin 24 SGPYSFGLamide
Procastatin 25 EDYDSSDQYSLamide
Procastatin 26 SGAYSFGLamide
Procastatin 3 QGMYSFGLamide
393
QGMYSFGL
>gi|140 |LiLab|LL140
PDMYSFGL
>gi|141 |LiLab|LL141
PDLYSFGL
>gi|142 |LiLab|LL142
ADMYSFGL
>gi|143 |LiLab|LL143
ADLYSFGL
>gi|144 |LiLab|LL144
SGNYNFGL
>gi|145 |LiLab|LL145
SRQYSFGL
>gi|146 |LiLab|LL146
PO
AGWNKFQGSW
>gi|147 |LiLab|LL147
AGWSSMRGAW
>gi|148 |LiLab|LL148
AGWSSTSRAW
>gi|149 |LiLab|LL149
AWSNLGQAW
>gi|150 |LiLab|LL150
GSNWSNLRGAW
>gi|151 |LiLab|LL151
GVNWSNLRGAW
>gi|152 |LiLab|LL152
LGNWNKFQGSW
>gi|153 |LiLab|LL153
LGNWSNLRGAW
>gi|154 |LiLab|LL154
LMFAPLAWPKGGARW
>gi|155 |LiLab|LL155
LNNNWSKFQGSW
>gi|156 |LiLab|LL156
LNWNKFQGSW
>gi|157 |LiLab|LL157
MFAPLAWPKGGARW
>gi|158 |LiLab|LL158
NDWSKFGQSW
>gi|159 |LiLab|LL159
NNNWTKFQGSW
>gi|160 |LiLab|LL160
NNWSGAFKGSW
>gi|161 |LiLab|LL161
NPDWAHFRGSW
>gi|162 |LiLab|LL162
SGDWSSLRGAW
>gi|163 |LiLab|LL163
SKWNKFQGSW
>gi|164 |LiLab|LL164
STDWSSLRSAW
>gi|165 |LiLab|LL165
TGWNKFQGSW
Allatostatin A-type Procastatin 4 PDMYSFGLamide
Allatostatin A-type Procastatin 5 PDLYSFGLamide
Allatostatin A-type Procastatin 6 ADMYSFGLamide
Allatostatin A-type Procastatin 7 ADLYSFGLamide
Allatostatin A-type Procastatin 8 SGNYNFGLamide
Allatostatin A-type Procastatin 9 SRQYSFGLamide
Allatostatin B-type
AGWNKFQGSWamide Csap PO Cmaen
Allatostatin B-type
AGWSSMRGAWamide Csap PO
Allatostatin B-type
AGWSSTSRAWamide Csap br
Allatostatin B-type
AWSNLGQAWamide Cmaen PO
Allatostatin B-type
GSNWSNLRGAWamide Cmaen PO
Allatostatin B-type
GVNWSNLRGAWamide Cmaen VNC/PO
Allatostatin B-type
LGNWNKFQGSWamide Csap
Allatostatin B-type
LGNWSNLRGAWamide Cb PO
Allatostatin B-type
LMFAPLAWPKGGARWamide Csap
Allatostatin B-type
LNNNWSKFQGSWamide Csap
Allatostatin B-type
LNWNKFQGSWamide Csap
Allatostatin B-type
MFAPLAWPKGGARWamide Csap PO
Allatostatin B-type
NDWSKFGQSWamide Csap
Allatostatin B-type
NNNWTKFQGSWamide Csap br
Allatostatin B-type
NNWSGAFKGSWamide Csap
Allatostatin B-type
NPDWAHFRGSWamide Csap
Allatostatin B-type
SGDWSSLRGAWamide Csap PO,br
Allatostatin B-type
SKWNKFQGSWamide Hoa.brain
Allatostatin B-type
STDWSSLRSAWamide Csap PO,br
Allatostatin B-type
TGWNKFQGSWamide Csap br
394
>gi|166 |LiLab|LL166 Allatostatin B-type TNWNKFQGSWamide Hoa. Brain
TNWNKFQGSW
>gi|167 |LiLab|LL167 Allatostatin B-type TQNWTKFQGSWamide Csap
TQNWTKFQGSW
>gi|168 |LiLab|LL168 Allatostatin B-type VTWGKFQGSWamide Cmaen PO
VTWGKFQGSW
>gi|169 |LiLab|LL169 Allatostatin B-type CbAST-B1 VPNDWAHFRGSWamide Csap
PO,br Cmaen Br/VNC/PO Cb br,PO,CoG
VPNDWAHFRGSW
>gi|170 |LiLab|LL170 Allatostatin B-type CbAST-B2 QWSSMRGAWamide Cmaen
Br/PO
QWSSMRGAW
>gi|171 |LiLab|LL171 Allatostatin B-type CbAST-B3 SGKWSNLRGAWamide Csap
PO Cmaen Br/PO
SGKWSNLRGAW
>gi|172 |LiLab|LL172 Allatostatin B-type CbAST-B4 NWNKFQGSWamide Csap PO
Cb br,PO,STG
NWNKFQGSW
>gi|173 |LiLab|LL173 Allatostatin B-type CbAST-B5 TSWGKFQGSWamide Csap
PO,br Cmaen PO Cb br,PO,STG
TSWGKFQGSW
>gi|174 |LiLab|LL174 Allatostatin B-type CbAST-B6 GNWNKFQGSWamide Csap
PO,br Cmaen PO Cb br,PO,STG,CoG
GNWNKFQGSW
>gi|175 |LiLab|LL175 Allatostatin B-type CbAST-B7 NNWSKFQGSWamide Csap
PO,br Cmaen PO Cb br,PO,STG,CoG
NNWSKFQGSW
>gi|176 |LiLab|LL176 Allatostatin B-type CbAST-B8 STNWSSLRSAWamide Csap
PO,br Cmaen VNC/PO Cb PO
STNWSSLRSAW
>gi|177 |LiLab|LL177 Allatostatin B-type CbAST-B9 NNNWSKFQGSWamide Csap
PO Cmaen Br/PO Cb br,PO,STG,CoG
NNNWSKFQGSW
>gi|178 |LiLab|LL178 Allatostatin B-type Lepidopteran peptide MIP IV
AWSALHGAWamide
AWSALHGAW
>gi|179 |LiLab|LL179 Allatostatin Combos DPYAFGLGKRPADL Cb.PO.
DPYAFGLGKRPADL
>gi|180 |LiLab|LL180 Allatostatin Combos DPYAFGLGKRPADLYEFGLamide Cb.PO.
DPYAFGLGKRPADLYEFGL
>gi|181 |LiLab|LL181 Allatostatin Combos DPYAFGLGKRPDMYAFGLamide Cb.PO.
DPYAFGLGKRPDMYAFGL
>gi|182 |LiLab|LL182 Allatostatin Combos DPYAFGLGKRPDMYGFGLamide Cb.PO.
DPYAFGLGKRPDMYGFGL
>gi|183 |LiLab|LL183 Allatostatin Combos EPYAFGLGKRPATDL Cb.PO.
EPYAFGLGKRPATDL
>gi|184 |LiLab|LL184 Allatostatin Combos
EPYAFGLGKRPATDLYAFGLamide
Cb.PO.
EPYAFGLGKRPATDLYAFGL
>gi|185 |LiLab|LL185 Allatostatin Combos
GSGQYAFGLGKKAGGAYSFGLamide
Cb.PO.
GSGQYAFGLGKKAGGAYSFGL
>gi|186 |LiLab|LL186 Allatostatin C-type
SYWKQCAFNAVSCFamide Cb cog,
hoa cog, capr cog, Cmaen cog
395
SYWKQCAFNAVSCF
>gi|187 |LiLab|LL187 Allatostatin C-type
PISCF-AST pQIRYHQcYFNPIScF Cb
PO, CoG, brain and Hoa. PO, CoG, brain
QIRYHQCYFNPISCF
>gi|188 |LiLab|LL188 Allatostatin C-type Lepidopteran peptide Manse-AS
pEVRFRQCYFNPISCF
QVRFRQCYFNPISCF
>gi|189
|LiLab|LL189
Allatotropin
Lepidopteran
peptide
Manse-AT
GFKNVEMMTARGFamide Cp.SG
GFKNVEMMTARGF
>gi|190
|LiLab|LL190
bursicon
bursicon
alpha
DECSLRPVIHILSYPGCTSKPIPSFACQGRCTSYVQVSGSKLWQTERSMCCQESGEREAAITLNCPKPRPGEP
KEKKVLTRAPIDCMCRPCTDVEEGTVLAQKIANFIQDSMPDSVPFLK
DECSLRPVIHILSYPGCTSKPIPSFACQGRCTSYVQVSGSKLWQTERSMCCQESGEREAAITLNCPKPRPGEP
KEKKVLTRAPIDCMCRPCTDVEEGTVLAQKIANFIQDSMPDSVPFLK
>gi|191
|LiLab|LL191
bursicon
bursicon
beta
RSYGECETLPSTIHISKEEYDDTGRLVRVCEEDVAVNKCEGACVSKVQPSVNTPSGFLKDCRCCREVHLRARD
ITLTHCYDGDGARLSGAKATQHVKLREPADCQCFKCGDSTR
RSYGECETLPSTIHISKEEYDDTGRLVRVCEEDVAVNKCEGACVSKVQPSVNTPSGFLKDCRCCREVHLRARD
ITLTHCYDGDGARLSGAKATQHVKLREPADCQCFKCGDSTR
>gi|192
|LiLab|LL192
Calcitonin-like
diuretic
hormone
GLDLGLGRGFSGSQAAKHLMGLAAANFANFAGGPamide
GLDLGLGRGFSGSQAAKHLMGLAAANFANFAGGP
>gi|193 |LiLab|LL193 CCAP CCAP PFCNAFTGCamide Csap SG,PO Cmaen Br/VNC/PO
Cb br,PO
PFCNAFTGC
>gi|194 |LiLab|LL194 CCAP CCAP precursor related peptide DIGDLLEGKD
DIGDLLEGKD
>gi|195 |LiLab|LL195 CCAP Lepidopteran peptide CAP2b pELYAFPRVamide
QLYAFPRV
>gi|196 |LiLab|LL196 CHH (Crustacean hyperglycemic hormone) CanboCHH
pQIYDTSCKGVYDRALFSDLEHVCDDCYNLYRSSYVASECRRNCYSNVVFRQCMEELLLLMEEFDKYARAVQI
Vamide Cb
QIYDTSCKGVYDRALFSDLEHVCDDCYNLYRSSYVASECRRNCYSNVVFRQCMEELLLLMEEFDKYARAVQIV
>gi|197 |LiLab|LL197 CHH (Crustacean hyperglycemic hormone) CanpaCHH-II
pEIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQVMEELLLMEEFDKYARAVQIV
amide Cancer pagarus
QIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQVMEELLLMEEFDKYARAVQIV
>gi|198 |LiLab|LL198 CHH (Crustacean hyperglycemic hormone) CanpaMIH
RVINDECPNLIGNRDLYKKVEWICEDCSNIFRKTGMASLCRRNCFFNEDFVWCVHATERSEELEDLEEWVGIL
GAGRD
RVINDECPNLIGNRDLYKKVEWICEDCSNIFRKTGMASLCRRNCFFNEDFVWCVHATERSEELEDLEEWVGIL
GAGRD
>gi|199 |LiLab|LL199 CHH (Crustacean hyperglycemic hormone) CanpaMOIH1
RRINNDCQNFIGNRAMYEKVDWICKDCANIFRKDGLLNNCRSNCFYNTEFLWCIDATENTRNKEQLEQWAAIL
GAGWN Cancer pagarus
RRINNDCQNFIGNRAMYEKVDWICKDCANIFRKDGLLNNCRSNCFYNTEFLWCIDATENTRNKEQLEQWAAIL
GAGWN
>gi|200 |LiLab|LL200 CHH (Crustacean hyperglycemic hormone) CanpaMOIH2
RRINNDCQNFIGNRAMYEKVDWICKDCANIFRQDGLLNNCRSNCFYNTEFLWCIDATENTRNKEQLEQWAAIL
GAGWN
RRINNDCQNFIGNRAMYEKVDWICKDCANIFRQDGLLNNCRSNCFYNTEFLWCIDATENTRNKEQLEQWAAIL
GAGWN
396
>gi|201 |LiLab|LL201 CHH (Crustacean hyperglycemic hormone) CanproCHH-II
QIYDSSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQVMEELLLMEEFDKYARAVQIVa
mide Cancer productus
QIYDSSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQVMEELLLMEEFDKYARAVQIV
>gi|202 |LiLab|LL202 CHH (Crustacean hyperglycemic hormone) Capa-CHH II
pEIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMDEFDKYARAVQIV
Cancer pagurus
QIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMDEFDKYARAVQIV
>gi|203 |LiLab|LL203 CHH (Crustacean hyperglycemic hormone) CapaMIH
RVINDDCPNLIGNRDLYKKVEWICEDCSNIFRNTGMATLCRKNCFFNEDFLWCVYATERTEEMSQLRQWVGIL
GAGRE
RVINDDCPNLIGNRDLYKKVEWICEDCSNIFRNTGMATLCRKNCFFNEDFLWCVYATERTEEMSQLRQWVGIL
GAGRE
>gi|204 |LiLab|LL204 CHH (Crustacean hyperglycemic hormone) Capr-CHH I
QIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIV
QIYDTSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIV
>gi|205 |LiLab|LL205 CHH (Crustacean hyperglycemic hormone) Capr-CHH II
QIYDSSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIV
QIYDSSCKGVYDRGLFSDLEHVCDDCYNLYRNSYVASACRSNCYSNVVFRQCMEELLLMEEFDKYARAVQIV
>gi|206 |LiLab|LL206 CHH (Crustacean hyperglycemic hormone) CarmaCHH
pQIYDTSCKGVYDRALFNDLEHVCDDCYNLYRTSYVASACRSNCYSNLVFRQCMDDLLMMDEFDQYARKVQMV
amide Cmaen
QIYDTSCKGVYDRALFNDLEHVCDDCYNLYRTSYVASACRSNCYSNLVFRQCMDDLLMMDEFDQYARKVQMV
>gi|207 |LiLab|LL207 CHH (Crustacean hyperglycemic hormone) CarmaMIH
RVINDDCPNLIGNRDLYKRVEWICEDCSNIFRNTGMATLCRKNCFFNEDFLWCVYATERTEEMSQLRQWVGIL
GAGRE
RVINDDCPNLIGNRDLYKRVEWICEDCSNIFRNTGMATLCRKNCFFNEDFLWCVYATERTEEMSQLRQWVGIL
GAGRE
>gi|208 |LiLab|LL208 CHH (Crustacean hyperglycemic hormone) CarmaPOCHH
pQIYDTSCKGVYDRALFNDLEHVCDDCYNLYRTSYVASACRNNCFENEVFDVCVYQLYFPNHEEYLRSRDGLK
G Cmaen PO
QIYDTSCKGVYDRALFNDLEHVCDDCYNLYRTSYVASACRNNCFENEVFDVCVYQLYFPNHEEYLRSRDGLKG
>gi|209 |LiLab|LL209 CHH (Crustacean hyperglycemic hormone) DappuITPL
SFFDIQCKGNYDKSIFARLDRICEDCYNLFREPQLHSLCRSDFKSPYFKGCLQALLLIDEEEKFNQMVEILam
ide Daphnia pulex
SFFDIQCKGNYDKSIFARLDRICEDCYNLFREPQLHSLCRSDFKSPYFKGCLQALLLIDEEEKFNQMVEIL
>gi|210 |LiLab|LL210 CHH (Crustacean hyperglycemic hormone) DappuITPL
SFFEDINCKGLYDKSIFARLDRICQDCYSLYREPELHTLCRKNCFTTNYFKGCLDALLINDEKDIQRVMKDIS
IIHQIPI Daphnia pulex
SFFEDINCKGLYDKSIFARLDRICQDCYSLYREPELHTLCRKNCFTTNYFKGCLDALLINDEKDIQRVMKDIS
IIHQIPI
>gi|211 |LiLab|LL211 CHH (Crustacean hyperglycemic hormone) Hoa-CHH-A
(pCHH-A[pQ61-V132amide])
pEVFDQACKGVYDRNLFKKLDRVCEDCYNLYRKPFVATTCRENCYSNWVFRQCLDDLLLSDVIDEYVSNVQMV
amide
QVFDQACKGVYDRNLFKKLDRVCEDCYNLYRKPFVATTCRENCYSNWVFRQCLDDLLLSDVIDEYVSNVQMV
>gi|212 |LiLab|LL212 CHH (Crustacean hyperglycemic hormone) Hoa-CHH-B
(pCHH-B[pQ61-V132amide])
pEVFDQACKGVYDRNLFKKLNRVCEDCYNLYRKPFIVTTCRENCYSNRVFRQCLDDLLMIDVIDEYVSNVQMV
amide
QVFDQACKGVYDRNLFKKLNRVCEDCYNLYRKPFIVTTCRENCYSNRVFRQCLDDLLMIDVIDEYVSNVQMV
>gi|213 |LiLab|LL213 CHH (Crustacean hyperglycemic hormone) Hoa-MIH
(pMIH[pQ61-M131])
pEVFDQACKGVYDRNLFKKLDRVCEDCYNLYRKPFVATTCRENCYSNWVFRQCLDDLLLSNVIDEYVSNVQM
397
QVFDQACKGVYDRNLFKKLDRVCEDCYNLYRKPFVATTCRENCYSNWVFRQCLDDLLLSNVIDEYVSNVQM
>gi|214 |LiLab|LL214 CHH (Crustacean hyperglycemic hormone) HoaVIH
ASAWFTNDECPGVMGNRDLYEKVAWVCNDCANIFRNNDVGVMCKKDCFHTMWFLWCVYATERHGEIDQFRKWV
SILR
ASAWFTNDECPGVMGNRDLYEKVAWVCNDCANIFRNNDVGVMCKKDCFHTMWFLWCVYATERHGEIDQFRKWV
SILR
>gi|215 |LiLab|LL215 CHH (Crustacean hyperglycemic hormone) HomamVIH
ASAWFTNDECPGVMGNRDLYEKVAWVCNDCANIFRNNDVGVMCKKDCFHTMDFLWCVYATERHGEIDQFRKWV
SILRAamide Hoa
ASAWFTNDECPGVMGNRDLYEKVAWVCNDCANIFRNNDVGVMCKKDCFHTMDFLWCVYATERHGEIDQFRKWV
SILRA
>gi|216 |LiLab|LL216 CHH (Crustacean hyperglycemic hormone) Mee-MIH
SYIENTCRGVMGNRDIYKKVVRVCEDCTNIFRLPGLDGMCRDRCFNNEWFLVCLKAANRDDELDKFKVWISIL
NPGL Metepenaeus ensis
SYIENTCRGVMGNRDIYKKVVRVCEDCTNIFRLPGLDGMCRDRCFNNEWFLVCLKAANRDDELDKFKVWISIL
NPGL
>gi|217 |LiLab|LL217 CHH (Crustacean hyperglycemic hormone) MetenCHHA
SLFDPSCSGVFDRELLGRLNRVCDDCYNVFRDPKVAMECKSNCFLNPAFIQCLEYLLPEDLHEEYQSHQVVam
ide Metapenaeus ensis
SLFDPSCSGVFDRELLGRLNRVCDDCYNVFRDPKVAMECKSNCFLNPAFIQCLEYLLPEDLHEEYQSHQVV
>gi|218 |LiLab|LL218 CHH (Crustacean hyperglycemic hormone) Orl-CHH
pEVFDQACKGIYDRAIFKKLDRVCEDCYNLYRKPYVATTCRQNCYANSVFRQCLDDLLLIDVLDEYISGVQTV
amide Orconectes limosus
QVFDQACKGIYDRAIFKKLDRVCEDCYNLYRKPYVATTCRQNCYANSVFRQCLDDLLLIDVLDEYISGVQTV
>gi|219 |LiLab|LL219 CHH (Crustacean hyperglycemic hormone) Pej-MIH
SFIDNRCRGVMGNRDIYKKVVRVCEDCTNIFRLGLDGMCRNRCFYNEWFLICLKAANREDEIEKFRVWISILN
AGQ M. japonicus
SFIDNRCRGVMGNRDIYKKVVRVCEDCTNIFRLGLDGMCRNRCFYNEWFLICLKAANREDEIEKFRVWISILN
AGQ
>gi|220 |LiLab|LL220 CHH (Crustacean hyperglycemic hormone) Prc-MIH
RYVFEECPGVMGNRAVHGKVTRVCEDCYNVFRDTDVLAGCRKGCFSSEMFKLCLLAMERVEEFPDFKRWIGLL
NAamide Procambarus clarkii
RYVFEECPGVMGNRAVHGKVTRVCEDCYNVFRDTDVLAGCRKGCFSSEMFKLCLLAMERVEEFPDFKRWIGLL
NA
>gi|221 |LiLab|LL221 Corazonin Corazonin pQTFQYSRGWTNamide Cmaen Br Cb
br,CoG
QTFQYSRGWTN
>gi|222 |LiLab|LL222 CPRP (CHH precursor-related peptide) (GLN-4)
RSTQGYGRMDRILAALKTSPMEPSAALAVEHGTTHPLE Cmaen SG/PO
RSTQGYGRMDRILAALKTSPMEPSAALAVEHGTTHPLE
>gi|223 |LiLab|LL223 CPRP (CHH precursor-related peptide) (PRO-4)
RSTPGYGRMDRILAALKTSPMEPSAALAVEHGTTHPLE Cmaen SG/PO
RSTPGYGRMDRILAALKTSPMEPSAALAVEHGTTHPLE
>gi|224 |LiLab|LL224 CPRP (CHH precursor-related peptide) ASLKSPTVTPLR
Csap SG
ASLKSPTVTPLR
>gi|225 |LiLab|LL225 CPRP (CHH precursor-related peptide) Bthe CPRP
RSAEGFGRMERLLASIRGGADSMGHLGELTGAGEGAGHPLE
RSAEGFGRMERLLASIRGGADSMGHLGELTGAGEGAGHPLE
>gi|226 |LiLab|LL226 CPRP (CHH precursor-related peptide) Cama CPRP I
RSTQGYGRMDRILAALKTSPMEPSAALAVENGTTHPLE
RSTQGYGRMDRILAALKTSPMEPSAALAVENGTTHPLE
>gi|227 |LiLab|LL227 CPRP (CHH precursor-related peptide) Cama CPRP II
RSTQGYGRMDRILAALKTSPMEPSAALAVQHGTTHPLE
398
RSTQGYGRMDRILAALKTSPMEPSAALAVQHGTTHPLE
>gi|228 |LiLab|LL228 CPRP (CHH precursor-related peptide) Capa CPRP
RSAQGMGKMERLLASYRGALEPSTPLGDLSGSLGHPVE
RSAQGMGKMERLLASYRGALEPSTPLGDLSGSLGHPVE
>gi|229 |LiLab|LL229 CPRP (CHH precursor-related peptide) Capr CPRP I
RSAQGMGKMEHLLASYRGALESNTPTGDLPGGLVHPVE Cp.SG
RSAQGMGKMEHLLASYRGALESNTPTGDLPGGLVHPVE
>gi|230 |LiLab|LL230 CPRP (CHH precursor-related peptide) Capr CPRP II
RSAQGMGKMERLLASYRGAVEPNTPLGDLPGGLVHPVE Cp.SG
RSAQGMGKMERLLASYRGAVEPNTPLGDLPGGLVHPVE
>gi|231 |LiLab|LL231 CPRP (CHH precursor-related peptide) Capr CPRP III
RSAQGMGKMEHLLASYRGALESNTPLGDLPGGLVHPVE Cp.SG
RSAQGMGKMEHLLASYRGALESNTPLGDLPGGLVHPVE
>gi|232 |LiLab|LL232 CPRP (CHH precursor-related peptide) Capr CPRP IV
RSAQGMGKMERLLASYRAAVEPNTPLGDLPGGLVHPVE Cp.SG
RSAQGMGKMERLLASYRAAVEPNTPLGDLPGGLVHPVE
>gi|233 |LiLab|LL233 CPRP (CHH precursor-related peptide) Casap CPRP
RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE Csap SG
RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE
>gi|234 |LiLab|LL234 CPRP (CHH precursor-related peptide) Cb CPRP I
RSAQGLGKMERLLASYRGALEPNTPLGDLSGSVGHPVE Cb.SG
RSAQGLGKMERLLASYRGALEPNTPLGDLSGSVGHPVE
>gi|235 |LiLab|LL235 CPRP (CHH precursor-related peptide) Cb CPRP II
RSAQGLGKMERLLASYRGALEPNTPLGDLSGSLGHPVE Cb.SG
RSAQGLGKMERLLASYRGALEPNTPLGDLSGSLGHPVE
>gi|236 |LiLab|LL236 CPRP (CHH precursor-related peptide) Cb CPRP III
RSAQGLGKMEHLLASYRGALEPNTPLGDLSGSLGHPVE Cb.SG
RSAQGLGKMEHLLASYRGALEPNTPLGDLSGSLGHPVE
>gi|237 |LiLab|LL237 CPRP (CHH precursor-related peptide) Cb CPRP IV
RSAQGLGKMERLLVSYRGAVEPNTPLGDLSGSLGHPVE Cb.SG
RSAQGLGKMERLLVSYRGAVEPNTPLGDLSGSLGHPVE
>gi|238 |LiLab|LL238 CPRP (CHH precursor-related peptide) DLKSDTVTPLR
Csap SG
DLKSDTVTPLR
>gi|239 |LiLab|LL239 CPRP (CHH precursor-related peptide) GFLSQDVHS
GFLSQDVHS
>gi|240 |LiLab|LL240 CPRP (CHH precursor-related peptide) Hoa CPRP A (I16) RSVEGASRMEKLLSSISPSSTPLGFLSQDHSVN Hoa.SG
RSVEGASRMEKLLSSISPSSTPLGFLSQDHSVN
>gi|241 |LiLab|LL241 CPRP (CHH precursor-related peptide) Hoa CPRP C
RSVEGVSRMEKLLSSISPSSMPLGFLSQDHSVN Hoa.SG
RSVEGVSRMEKLLSSISPSSMPLGFLSQDHSVN
>gi|242 |LiLab|LL242 CPRP (CHH precursor-related peptide) Hoa CPRP-A (SN16) RSVEGASRMEKLLSSSNSPSSTPLGFLSQDHSVN Hoa.SG
RSVEGASRMEKLLSSSNSPSSTPLGFLSQDHSVN
>gi|243 |LiLab|LL243 CPRP (CHH precursor-related peptide) Hoa CPRP-B
RSVEGVSRMEKLLSSISPSSTPLGFLSQDHSVN Hoa.SG
RSVEGVSRMEKLLSSISPSSTPLGFLSQDHSVN
>gi|244 |LiLab|LL244 CPRP (CHH precursor-related peptide) Mlan CPRP
WSLDGLARIEKLLSTSSSASAASPTRGQALNL
WSLDGLARIEKLLSTSSSASAASPTRGQALNL
>gi|245 |LiLab|LL245 CPRP (CHH precursor-related peptide) Mros CPRP
WSVDGLARIEKLLSTSSSASAASPTRGQALNL
WSVDGLARIEKLLSTSSSASAASPTRGQALNL
399
>gi|246 |LiLab|LL246 CPRP (CHH precursor-related peptide) Nnor CPRP A
RSVEGASRMEKLLSSSNSPSSTPLGFLSQEHSVN
RSVEGASRMEKLLSSSNSPSSTPLGFLSQEHSVN
>gi|247 |LiLab|LL247 CPRP (CHH precursor-related peptide) Nnor CPRP B
RSVEGASRMEKLLSSISPSSTPLGFLSQEHSVN
RSVEGASRMEKLLSSISPSSTPLGFLSQEHSVN
>gi|248 |LiLab|LL248 CPRP (CHH precursor-related peptide) Olim CPRP A
RSVEGSSRMERLLSSGSSSSEPLSFLSQDHSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDHSVS
>gi|249 |LiLab|LL249 CPRP (CHH precursor-related peptide) Olim CPRP A*
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
>gi|250 |LiLab|LL250 CPRP (CHH precursor-related peptide) Olim CPRP B,
Pcla CPRP RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
>gi|251
|LiLab|LL251
CPRP
(CHH
precursor-related
peptide)
PSAALAVEHGTTHPLE Cmaen SG
PSAALAVEHGTTHPLE
>gi|252
|LiLab|LL252
CPRP
(CHH
precursor-related
peptide)
RASQGLGKMERLLASRGALEPN Cb SG
RASQGLGKMERLLASRGALEPN
>gi|253 |LiLab|LL253 CPRP (CHH precursor-related peptide) RGFEGETGHPN
Csap SG
RGFEGETGHPN
>gi|254 |LiLab|LL254 CPRP (CHH precursor-related peptide) RSAEGLGRMamide
Csap SG
RSAEGLGRM
>gi|255 |LiLab|LL255 CPRP (CHH precursor-related peptide) RSAEGLGRMG Csap
SG
RSAEGLGRMG
>gi|256 |LiLab|LL256 CPRP (CHH precursor-related peptide) RSAEGLGRMGRL
Csap SG
RSAEGLGRMGRL
>gi|257 |LiLab|LL257 CPRP (CHH precursor-related peptide) RSAEGLGRMGRLL
Csap SG
RSAEGLGRMGRLL
>gi|258 |LiLab|LL258 CPRP (CHH precursor-related peptide) RSAEGLGRVGRLL
Csap SG
RSAEGLGRVGRLL
>gi|259 |LiLab|LL259 CPRP (CHH precursor-related peptide) RSAQGLGKM(O)ERL
Cb PO,CoG
RSAQGLGKMERL
>gi|260
|LiLab|LL260
CPRP
(CHH
precursor-related
peptide)
RSAQGLGKMEHLLASY Cb SG
RSAQGLGKMEHLLASY
>gi|261
|LiLab|LL261
CPRP
(CHH
precursor-related
peptide)
RSAQGLGKYLRLLASY Cb SG
RSAQGLGKYLRLLASY
>gi|262 |LiLab|LL262 CPRP (CHH precursor-related peptide) RSVEGASRMEKLLT
RSVEGASRMEKLLT
>gi|263 |LiLab|LL263 CPRP (CHH precursor-related peptide) RSVEGVSRMEKLLT
RSVEGVSRMEKLLT
>gi|264 |LiLab|LL264 CPRP (CHH precursor-related peptide) SLKSDTVTPLLG
Csap SG
400
SLKSDTVTPLLG
>gi|265 |LiLab|LL265 CPRP (CHH precursor-related peptide) Sserr CPRP
RSAEGFGRMGRLLASLKADSLGPVQDFGVEGAAHPVE
RSAEGFGRMGRLLASLKADSLGPVQDFGVEGAAHPVE
>gi|266 |LiLab|LL266 CPRP (CHH precursor-related peptide) truncated (PRO4) 1-13 RSTPGYGRMDRIL Cmaen SG
RSTPGYGRMDRIL
>gi|267 |LiLab|LL267 CPRP (CHH precursor-related peptide) truncated (PRO4) 1-15 RSTPGYGRMDRILAA Cmaen SG
RSTPGYGRMDRILAA
>gi|268 |LiLab|LL268 CPRP (CHH precursor-related peptide) truncated (PRO4) 18-38 TSPMEPSAALAVEHGTTHPLE Cmaen SG
TSPMEPSAALAVEHGTTHPLE
>gi|269 |LiLab|LL269 CPRP (CHH precursor-related peptide) truncated (PRO4) 19-38 SPMEPSAALAVEHGTTHPLE Cmaen SG
SPMEPSAALAVEHGTTHPLE
>gi|270 |LiLab|LL270 CPRP (CHH precursor-related peptide) truncated Cama
CPRP I 1-13 RSTQGYGRMDPIL Cmaen SG
RSTQGYGRMDPIL
>gi|271 |LiLab|LL271 CPRP (CHH precursor-related peptide) truncated Cama
CPRP I 1-15 RSTQGYGRMDRILAA Cmaen SG
RSTQGYGRMDRILAA
>gi|272 |LiLab|LL272 CPRP (CHH precursor-related peptide) truncated Capa
CPRP 1-12, Capr CPRP II 1-12 RSAQGMGKMERL Cp.SG & PO
RSAQGMGKMERL
>gi|273 |LiLab|LL273 CPRP (CHH precursor-related peptide) truncated Capa
CPRP 24-38, CbCPRP II,III&IV 24-38 TPLGDLSGSLGHPVE Cb SG
TPLGDLSGSLGHPVE
>gi|274 |LiLab|LL274 CPRP (CHH precursor-related peptide) truncated Capr
CPRP I 1-12 RSAQGMGKMEHL Cp.SG & PO
RSAQGMGKMEHL
>gi|275 |LiLab|LL275 CPRP (CHH precursor-related peptide) truncated Capr
CPRP I 1-13 RSAQGMGKMEHLL Cp.SG & PO
RSAQGMGKMEHLL
>gi|276 |LiLab|LL276 CPRP (CHH precursor-related peptide) truncated Capr
CPRP I 1-14 RSAQGMGKMEHLLA Cp.SG & PO
RSAQGMGKMEHLLA
>gi|277 |LiLab|LL277 CPRP (CHH precursor-related peptide) Truncated Capr
CPRP I 30-38 PGGLVHPVE Cp.SG & PO
PGGLVHPVE
>gi|278 |LiLab|LL278 CPRP (CHH precursor-related peptide) Truncated Capr
CPRP II [13-38] LASYRGAVEPNTPLGDLPGGLVHPVE Cp.SG & PO
LASYRGAVEPNTPLGDLPGGLVHPVE
>gi|279 |LiLab|LL279 CPRP (CHH precursor-related peptide) truncated Casap
CPRP 13-25 LASLKSDTVTPLR Csap SG
LASLKSDTVTPLR
>gi|280 |LiLab|LL280 CPRP (CHH precursor-related peptide) truncated Casap
CPRP 14-25 ASLKSDTVTPLR Csap SG
ASLKSDTVTPLR
>gi|281 |LiLab|LL281 CPRP (CHH precursor-related peptide) truncated Casap
CPRP 15-25 SLKSDTVTPLR Csap SG
SLKSDTVTPLR
>gi|282 |LiLab|LL282 CPRP (CHH precursor-related peptide) truncated Casap
CPRP 18-28 SDTVTPLRGFE Csap SG
401
SDTVTPLRGFE
>gi|283 |LiLab|LL283 CPRP (CHH precursor-related peptide) truncated Casap
CPRP 22-36 TPLRGFEGETGHPLE Csap SG
TPLRGFEGETGHPLE
>gi|284 |LiLab|LL284 CPRP (CHH precursor-related peptide) truncated Cb
CPRP I&II 1-14 RSAQGLGKMERLLA Cb SG
RSAQGLGKMERLLA
>gi|285 |LiLab|LL285 CPRP (CHH precursor-related peptide) truncated Cb
CPRP I&II 1-15 RSAQGLGKMERLLAS Cb SG
RSAQGLGKMERLLAS
>gi|286 |LiLab|LL286 CPRP (CHH precursor-related peptide) truncated Cb
CPRP I&II 1-16 RSAQGLGKMERLLASY
RSAQGLGKMERLLASY
>gi|287 |LiLab|LL287 CPRP (CHH precursor-related peptide) truncated Cb
CPRP I,II&IV 1-12 RSAQGLGKMER Cb SG
RSAQGLGKMER
>gi|288 |LiLab|LL288 CPRP (CHH precursor-related peptide) truncated Cb
CPRP I,II&IV 1-13 RSAQGLGKMERLL Cb SG
RSAQGLGKMERLL
>gi|289 |LiLab|LL289 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II 18-38 GALEPNTPLGDLSGSLGHPVE Cb SG
GALEPNTPLGDLSGSLGHPVE
>gi|290 |LiLab|LL290 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II 2-38 SAQGLGKMERLLASYRGALEPNTPLGDLSGSLGHPVE
SAQGLGKMERLLASYRGALEPNTPLGDLSGSLGHPVE
>gi|291 |LiLab|LL291 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II 3-39 AQGLGKMERLLASYRGALEPNTPLGDLSGSLGHPVE
AQGLGKMERLLASYRGALEPNTPLGDLSGSLGHPVE
>gi|292 |LiLab|LL292 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II&III 12-38 LLASYRGALEPNTPLGDLSGSLGHPVE
LLASYRGALEPNTPLGDLSGSLGHPVE
>gi|293 |LiLab|LL293 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II&III 13-38 LASYRGALEPNTPLGDLSGSLGHPVE
LASYRGALEPNTPLGDLSGSLGHPVE
>gi|294 |LiLab|LL294 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II&III 16-38 RGALEPNTPLGDLSGSLGHPVE
RGALEPNTPLGDLSGSLGHPVE
>gi|295 |LiLab|LL295 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II&III 17-38 YRGALEPNTPLGDLSGSLGHPVE
YRGALEPNTPLGDLSGSLGHPVE
>gi|296 |LiLab|LL296 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II&III 19-38 ALEPNTPLGDLSGSLGHPVE
ALEPNTPLGDLSGSLGHPVE
>gi|297 |LiLab|LL297 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II,III&IV 23-38 NTPLGDLSGSLGHPVE
NTPLGDLSGSLGHPVE
>gi|298 |LiLab|LL298 CPRP (CHH precursor-related peptide) truncated Cb
CPRP II,III&IV15-38 PLGDLSGSLGHPVE
PLGDLSGSLGHPVE
>gi|299 |LiLab|LL299 CPRP (CHH precursor-related peptide) truncated Cb
CPRP IV 1-16 RSAQGLGKMERLLVSY
RSAQGLGKMERLLVSY
>gi|300 |LiLab|LL300 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-A (SN-16) 11-23 KLLSSSNSPSSTP
402
KLLSSSNSPSSTP
>gi|301 |LiLab|LL301 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-24 KLLSSSNSPSSTPL
KLLSSSNSPSSTPL
>gi|302 |LiLab|LL302 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-25 KLLSSSNSPSSTPLG
KLLSSSNSPSSTPLG
>gi|303 |LiLab|LL303 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-26 KLLSSSNSPSSTPLGF
KLLSSSNSPSSTPLGF
>gi|304 |LiLab|LL304 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-27 KLLSSSNSPSSTPLGFL
KLLSSSNSPSSTPLGFL
>gi|305 |LiLab|LL305 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-32 KLLSSSNSPSSTPLGFLSQDHS
KLLSSSNSPSSTPLGFLSQDHS
>gi|306 |LiLab|LL306 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-33 KLLSSSNSPSSTPLGFLSQDHSV
KLLSSSNSPSSTPLGFLSQDHSV
>gi|307 |LiLab|LL307 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 11-34 KLLSSSNSPSSTPLGFLSQDHSVN
KLLSSSNSPSSTPLGFLSQDHSVN
>gi|308 |LiLab|LL308 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-20 RSVEGASRMEKLLSSSNSPS
RSVEGASRMEKLLSSSNSPS
>gi|309 |LiLab|LL309 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 12-24 LLSSSNSPSSTPL
LLSSSNSPSSTPL
>gi|310 |LiLab|LL310 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 12-33 LLSSSNSPSSTPLGFLSQDHSV
LLSSSNSPSSTPLGFLSQDHSV
>gi|311 |LiLab|LL311 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 12-34 LLSSSNSPSSTPLGFLSQDHSVN
LLSSSNSPSSTPLGFLSQDHSVN
>gi|312 |LiLab|LL312 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-25 RSVEGASRMEKLLSSSNSPSSTPLG
RSVEGASRMEKLLSSSNSPSSTPLG
>gi|313 |LiLab|LL313 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-26 RSVEGASRMEKLLSSSNSPSSTPLGF
RSVEGASRMEKLLSSSNSPSSTPLGF
>gi|314 |LiLab|LL314 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-27 RSVEGASRMEKLLSSSNSPSSTPLGFL
RSVEGASRMEKLLSSSNSPSSTPLGFL
>gi|315 |LiLab|LL315 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-29 RSVEGASRMEKLLSSSNSPSSTPLGFLSQ
RSVEGASRMEKLLSSSNSPSSTPLGFLSQ
>gi|316 |LiLab|LL316 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-32 RSVEGASRMEKLLSSSNSPSSTPLGFLSQDHS
RSVEGASRMEKLLSSSNSPSSTPLGFLSQDHS
>gi|317 |LiLab|LL317 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 13-25 GFLSQDHSVN
GFLSQDHSVN
>gi|318 |LiLab|LL318 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 13-25 LSSSNSPSSTPLG
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
Truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
Truncated Hoa
Truncated Hoa
Truncated Hoa
Truncated Hoa
Truncated Hoa
truncated Hoa
truncated Hoa
403
LSSSNSPSSTPLG
>gi|319 |LiLab|LL319 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 1-33 RSVEGASRMEKLLSSSNSPSSTPLGFLSQDHSV
RSVEGASRMEKLLSSSNSPSSTPLGFLSQDHSV
>gi|320 |LiLab|LL320 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 13-32 LSSSNSPSSTPLGFLSQDHS
LSSSNSPSSTPLGFLSQDHS
>gi|321 |LiLab|LL321 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 13-33 LSSSNSPSSTPLGFLSQDHSV
LSSSNSPSSTPLGFLSQDHSV
>gi|322 |LiLab|LL322 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 13-34 LSSSNSPSSTPLGFLSQDHSVN
LSSSNSPSSTPLGFLSQDHSVN
>gi|323 |LiLab|LL323 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 14-33 SSSNSPSSTPLGFLSQDHSV
SSSNSPSSTPLGFLSQDHSV
>gi|324 |LiLab|LL324 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 14-34 SSSNSPSSTPLGFLSQDHSVN
SSSNSPSSTPLGFLSQDHSVN
>gi|325 |LiLab|LL325 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 15-32 SSNSPSSTPLGFLSQDHS Hoa.SG
SSNSPSSTPLGFLSQDHS
>gi|326 |LiLab|LL326 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 15-33 SSNSPSSTPLGFLSQDHSV
SSNSPSSTPLGFLSQDHSV
>gi|327 |LiLab|LL327 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 15-34 SSNSPSSTPLGFLSQDHSVN
SSNSPSSTPLGFLSQDHSVN
>gi|328 |LiLab|LL328 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 16-33 SNSPSSTPLGFLSQDHSV
SNSPSSTPLGFLSQDHSV
>gi|329 |LiLab|LL329 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 17-32 NSPSSTPLGFLSQDHS
NSPSSTPLGFLSQDHS
>gi|330 |LiLab|LL330 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 17-34 NSPSSTPLGFLSQDHSVN
NSPSSTPLGFLSQDHSVN
>gi|331 |LiLab|LL331 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 19-34, B 18-33 PSSTPLGFLSQDHSVN
PSSTPLGFLSQDHSVN
>gi|332 |LiLab|LL332 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 22-33, B 21-32 TPLGFLSQDHSV
TPLGFLSQDHSV
>gi|333 |LiLab|LL333 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 22-34, B 21-33 TPLGFLSQDHSVN
TPLGFLSQDHSVN
>gi|334 |LiLab|LL334 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 23-32, B&C 22-31 PLGFLSQDHS
PLGFLSQDHS
>gi|335 |LiLab|LL335 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 23-33, B&C 22-32 PLGFLSQDHSV
PLGFLSQDHSV
>gi|336 |LiLab|LL336 CPRP (CHH precursor-related peptide)
CPRP-A (SN-16) 23-34, B&C 22-33 PLGFLSQDHSVN
Truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
truncated Hoa
404
PLGFLSQDHSVN
>gi|337 |LiLab|LL337 CPRP (CHH precursor-related
CPRP-A (SN-16) 24-34, B&C 23-33 LGFLSQDHSVN
LGFLSQDHSVN
>gi|338 |LiLab|LL338 CPRP (CHH precursor-related
CPRP-A (SN-16) 26-33 FLSQDHSV
FLSQDHSV
>gi|339 |LiLab|LL339 CPRP (CHH precursor-related
CPRP-A (SN-16) 26-34 FLSQDHSVN
FLSQDHSVN
>gi|340 |LiLab|LL340 CPRP (CHH precursor-related
CPRP-A (SN-16) 9-33 MEKLLSSSNSPSSTPLGFLSQDHSV
MEKLLSSSNSPSSTPLGFLSQDHSV
>gi|341 |LiLab|LL341 CPRP (CHH precursor-related
CPRP-A (SN-16) 9-34 MEKLLSSSNSPSSTPLGFLSQDHSVN
MEKLLSSSNSPSSTPLGFLSQDHSVN
>gi|342 |LiLab|LL342 CPRP (CHH precursor-related
CPRP-A (SN-16, I-16) 1-12 RSVEGASRMEKL
RSVEGASRMEKL
>gi|343 |LiLab|LL343 CPRP (CHH precursor-related
CPRP-A (SN-16, I-16) 1-13 RSVEGASRMEKLL
RSVEGASRMEKLL
>gi|344 |LiLab|LL344 CPRP (CHH precursor-related
CPRP-A (SN-16, I-16) 1-14 RSVEGASRMEKLLS
RSVEGASRMEKLLS
>gi|345 |LiLab|LL345 CPRP (CHH precursor-related
CPRP-A (SN-16, I-16) 1-15 RSVEGASRMEKLLSS
RSVEGASRMEKLLSS
>gi|346 |LiLab|LL346 CPRP (CHH precursor-related
CPRP-A20-32, B19-31 SSTPLGFLSQDHS
SSTPLGFLSQDHS
>gi|347 |LiLab|LL347 CPRP (CHH precursor-related
CPRP-A20-34, B19-33 SSTPLGFLSQDHSVN
SSTPLGFLSQDHSVN
>gi|348 |LiLab|LL348 CPRP (CHH precursor-related
CPRP-A21-32, B20-31 STPLGFLSQDHS
STPLGFLSQDHS
>gi|349 |LiLab|LL349 CPRP (CHH precursor-related
CPRP-A21-34, B20-33 STPLGFLSQDHSVN
STPLGFLSQDHSVN
>gi|350 |LiLab|LL350 CPRP (CHH precursor-related
CPRP-B 11-23 KLLSSISPSSTPL
KLLSSISPSSTPL
>gi|351 |LiLab|LL351 CPRP (CHH precursor-related
CPRP-B 11-25 KLLSSISPSSTPLGF
KLLSSISPSSTPLGF
>gi|352 |LiLab|LL352 CPRP (CHH precursor-related
CPRP-B 11-26 KLLSSISPSSTPLGFL
KLLSSISPSSTPLGFL
>gi|353 |LiLab|LL353 CPRP (CHH precursor-related
CPRP-B 11-28 KLLSSISPSSTPLGFLSQ
KLLSSISPSSTPLGFLSQ
>gi|354 |LiLab|LL354 CPRP (CHH precursor-related
CPRP-B 11-32 KLLSSISPSSTPLGFLSQDHSV
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) Truncated Hoa
peptide) Truncated Hoa
peptide) Truncated Hoa
peptide) Truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
405
KLLSSISPSSTPLGFLSQDHSV
>gi|355 |LiLab|LL355 CPRP (CHH precursor-related
CPRP-B 11-33 KLLSSISPSSTPLGFLSQDHSVN
KLLSSISPSSTPLGFLSQDHSVN
>gi|356 |LiLab|LL356 CPRP (CHH precursor-related
CPRP-B 12-32 LLSSISPSSTPLGFLSQDHSV
LLSSISPSSTPLGFLSQDHSV
>gi|357 |LiLab|LL357 CPRP (CHH precursor-related
CPRP-B 12-33 LLSSISPSSTPLGFLSQDHSVN
LLSSISPSSTPLGFLSQDHSVN
>gi|358 |LiLab|LL358 CPRP (CHH precursor-related
CPRP-B 1-24 RSVEGVSRMEKLLSSISPSSTPLG
RSVEGVSRMEKLLSSISPSSTPLG
>gi|359 |LiLab|LL359 CPRP (CHH precursor-related
CPRP-B 1-26 RSVEGVSRMEKLLSSISPSSTPLGFL
RSVEGVSRMEKLLSSISPSSTPLGFL
>gi|360 |LiLab|LL360 CPRP (CHH precursor-related
CPRP-B 1-28 RSVEGVSRMEKLLSSISPSSTPLGFLSQ
RSVEGVSRMEKLLSSISPSSTPLGFLSQ
>gi|361 |LiLab|LL361 CPRP (CHH precursor-related
CPRP-B 1-32 RSVEGVSRMEKLLSSISPSSTPLGFLSQDHSV
RSVEGVSRMEKLLSSISPSSTPLGFLSQDHSV
>gi|362 |LiLab|LL362 CPRP (CHH precursor-related
CPRP-B 13-31 SISPSSTPLGFLSQDHS
SISPSSTPLGFLSQDHS
>gi|363 |LiLab|LL363 CPRP (CHH precursor-related
CPRP-B 13-32 LSSISPSSTPLGFLSQDHSV
LSSISPSSTPLGFLSQDHSV
>gi|364 |LiLab|LL364 CPRP (CHH precursor-related
CPRP-B 13-32 SISPSSTPLGFLSQDHSV
SISPSSTPLGFLSQDHSV
>gi|365 |LiLab|LL365 CPRP (CHH precursor-related
CPRP-B 13-33 LSSISPSSTPLGFLSQDHSVN
LSSISPSSTPLGFLSQDHSVN
>gi|366 |LiLab|LL366 CPRP (CHH precursor-related
CPRP-B 13-33 SISPSSTPLGFLSQDHSVN
SISPSSTPLGFLSQDHSVN
>gi|367 |LiLab|LL367 CPRP (CHH precursor-related
CPRP-B 14-31 SSISPSSTPLGFLSQDHS
SSISPSSTPLGFLSQDHS
>gi|368 |LiLab|LL368 CPRP (CHH precursor-related
CPRP-B 14-32 SSISPSSTPLGFLSQDHSV
SSISPSSTPLGFLSQDHSV
>gi|369 |LiLab|LL369 CPRP (CHH precursor-related
CPRP-B 14-33 SSISPSSTPLGFLSQDHSVN
SSISPSSTPLGFLSQDHSVN
>gi|370 |LiLab|LL370 CPRP (CHH precursor-related
CPRP-B 2-12 SVEGVSRMEKL
SVEGVSRMEKL
>gi|371 |LiLab|LL371 CPRP (CHH precursor-related
CPRP-B 2-33 SVEGVSRMEKLLSSISPSSTPLGFLSQDHSVN
SVEGVSRMEKLLSSISPSSTPLGFLSQDHSVN
>gi|372 |LiLab|LL372 CPRP (CHH precursor-related
CPRP-B 9-33 EKLLSSISPSSTPLGFLSQDHSVN
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
peptide) truncated Hoa
406
EKLLSSISPSSTPLGFLSQDHSVN
>gi|373 |LiLab|LL373 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-B&C 1-10 RSVEGVSRME
RSVEGVSRME
>gi|374 |LiLab|LL374 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-B&C 1-12 RSVEGVSRMEKL
RSVEGVSRMEKL
>gi|375 |LiLab|LL375 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-B&C 1-14 RSVEGVSRMEKLLS
RSVEGVSRMEKLLS
>gi|376 |LiLab|LL376 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-B&C 1-19 RSVEGVSRMEKLLSSISPS
RSVEGVSRMEKLLSSISPS
>gi|377 |LiLab|LL377 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 11-23 KLLSSISPSSMPLG
KLLSSISPSSMPLG
>gi|378 |LiLab|LL378 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 11-33 KLLSSISPSSMPLGFLSQDHSVN
KLLSSISPSSMPLGFLSQDHSVN
>gi|379 |LiLab|LL379 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 1-23 RSVEGVSRMEKLLSSISPSSMPL
RSVEGVSRMEKLLSSISPSSMPL
>gi|380 |LiLab|LL380 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 1-24 RSVEGVSRMEKLLSSISPSSMPLG
RSVEGVSRMEKLLSSISPSSMPLG
>gi|381 |LiLab|LL381 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 1-32 RSVEGVSRMEKLLSSISPSSMPLGFLSQDHSV
RSVEGVSRMEKLLSSISPSSMPLGFLSQDHSV
>gi|382 |LiLab|LL382 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 16-32 ISPSSMPLGFLSQDHSV
ISPSSMPLGFLSQDHSV
>gi|383 |LiLab|LL383 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 16-33 ISPSSMPLGFLSQDHSVN
ISPSSMPLGFLSQDHSVN
>gi|384 |LiLab|LL384 CPRP (CHH precursor-related peptide) truncated Hoa
CPRP-C 20-33 SMPLGFLSQDHSVN
SMPLGFLSQDHSVN
>gi|385
|LiLab|LL385
Ecdysis
triggering
hormone
ETH
DPSPEPFNPNYNRFRQKIPRIamide
DPSPEPFNPNYNRFRQKIPRI
>gi|386
|LiLab|LL386
Eclosion
hormone
AVAANRKVSICIKNCGQCKKMYTDYFNGGLCGDFCLQTEGRFIPDCNRPDILIPFFLQRLE
AVAANRKVSICIKNCGQCKKMYTDYFNGGLCGDFCLQTEGRFIPDCNRPDILIPFFLQRLE
>gi|387 |LiLab|LL387 Kinins cholecyctokinin cck8 DYMGWMDFamide
DYMGWMDF
>gi|388
|LiLab|LL388
Kinins
Lepidopteran
peptide
helicokinin
I
YFSPWGamide
YFSPWG
>gi|389
|LiLab|LL389
Kinins
Lepidopteran
peptide
helicokinin
II
VRFSPWGamide
VRFSPWG
>gi|390
|LiLab|LL390
Kinins
Lepidopteran
peptide
helicokinin
III
KVKFSAWGamide
KVKFSAWG
407
>gi|391 |LiLab|LL391 Kinins pev-Kinin 1 ASFSPWGamide
ASFSPWG
>gi|392 |LiLab|LL392 Kinins pev-Kinin 2 DFSAWAamide Cp.PO.
DFSAWA
>gi|393 |LiLab|LL393 Kinins pev-Kinin 3 PAFSPWGamide
PAFSPWG
>gi|394 |LiLab|LL394 Kinins pev-Kinin 4 VAFSPWGamide
VAFSPWG
>gi|395 |LiLab|LL395 Kinins pev-Kinin 5 pEAFSPWAamide
QAFSPWA
>gi|396 |LiLab|LL396 Kinins pev-Kinin 6 AFSPWAamide
AFSPWA
>gi|397 |LiLab|LL397 myosuppressin pQDLDHVFLR Csap PO
QDLDHVFLR
>gi|398
|LiLab|LL398
Neuroparsin
APRCDRHDEEAPKNCKYGTTQDWCKNGVCAKGPGETCGGYRWSEGKCGEGTPCSCGICGGCSPFDGKCGPTSI
C
APRCDRHDEEAPKNCKYGTTQDWCKNGVCAKGPGETCGGYRWSEGKCGEGTPCSCGICGGCSPFDGKCGPTSI
C
>gi|399 |LiLab|LL399 Orcokinin DEIDRSGFGFA Cp.SG Csap PO
DEIDRSGFGFA
>gi|400 |LiLab|LL400 Orcokinin DFDEIDRSGFA Csap SG Cb br,PO,SG
DFDEIDRSGFA
>gi|401 |LiLab|LL401 Orcokinin DFDEIDRSGFG Csap SG
DFDEIDRSGFG
>gi|402 |LiLab|LL402 Orcokinin DFDEIDRSGFGFA Csap SG
DFDEIDRSGFGFA
>gi|403 |LiLab|LL403 Orcokinin DFDEIDRSGFGFV Csap SG
DFDEIDRSGFGFV
>gi|404 |LiLab|LL404 Orcokinin DFDEIDRSSFA Csap SG,PO
DFDEIDRSSFA
>gi|405 |LiLab|LL405 Orcokinin DFDEIDRSSFG Csap SG
DFDEIDRSSFG
>gi|406 |LiLab|LL406 Orcokinin DFDEIDRSSFGFA Csap SG
DFDEIDRSSFGFA
>gi|407 |LiLab|LL407 Orcokinin DFDEIDRSSFGFN Csap SG
DFDEIDRSSFGFN
>gi|408 |LiLab|LL408 Orcokinin DFDEIDRSSFGFV
DFDEIDRSSFGFV
>gi|409 |LiLab|LL409 Orcokinin DFEDIERSGFGFV Cmaen VNC/PO
DFEDIERSGFGFV
>gi|410 |LiLab|LL410 Orcokinin EIDRSGFGF Csap SG
EIDRSGFGF
>gi|411 |LiLab|LL411 Orcokinin
EIDRSGFGFA Csap SG,PO Cmaen PO Cb
br,STG,CoG
EIDRSGFGFA
>gi|412 |LiLab|LL412 Orcokinin EIDRSSFGFN Cp.SG
EIDRSSFGFN
>gi|413 |LiLab|LL413 Orcokinin FDEIDRSGFA Csap SG
FDEIDRSGFA
>gi|414 |LiLab|LL414 Orcokinin FDEIDRSGFG Csap SG
FDEIDRSGFG
>gi|415 |LiLab|LL415 Orcokinin FDEIDRSGFGFA Csap PO Cmaen PO
FDEIDRSGFGFA
408
>gi|416 |LiLab|LL416 Orcokinin FDEIDRSSFA Csap SG
FDEIDRSSFA
>gi|417 |LiLab|LL417 Orcokinin NFDEIDRSA Csap SG
NFDEIDRSA
>gi|418 |LiLab|LL418 Orcokinin NFDEIDRSGFA Csap SG,PO,br Cmaen VNC/PO Cb
br,Sg,STG,CoG
NFDEIDRSGFA
>gi|419 |LiLab|LL419 Orcokinin
NFDEIDRSGFamide Hoa. SG/CNS Csap PO,br
Cmaen Br Cb br,SG,CoG
NFDEIDRSGF
>gi|420 |LiLab|LL420 Orcokinin NFDEIDRSGFGFH Hoa. SG/CNS
NFDEIDRSGFGFH
>gi|421 |LiLab|LL421 Orcokinin NFDEIDRSSFamide Csap SG,PO,br Cmaen Br
NFDEIDRSSF
>gi|422 |LiLab|LL422 Orcokinin NFDEIDRSSFG Csap SG,PO Cmaen VNC
NFDEIDRSSFG
>gi|423 |LiLab|LL423 Orcokinin NFDEIDRSSFGF Csap SG,PO,br
NFDEIDRSSFGF
>gi|424 |LiLab|LL424 Orcokinin NFDEIDRSSFGFA Csap br
NFDEIDRSSFGFA
>gi|425 |LiLab|LL425 Orcokinin SSEDMDRLGFA Hoa. SG/CNS
SSEDMDRLGFA
>gi|426 |LiLab|LL426 Orcokinin VYGPRDIANLY Hoa. SG/CNS
VYGPRDIANLY
>gi|427 |LiLab|LL427 Orcokinin [Ala13]-orcokinin NFDEIDRSGFGFA Csap
SG,PO,br Cmaen Br/VNC/SG/PO Cb br,PO,SG,STG,CoG
NFDEIDRSGFGFA
>gi|428 |LiLab|LL428 Orcokinin [Ala8-Ala13]-Orcokinin NFDEIDRAGFGFA
NFDEIDRAGFGFA
>gi|429 |LiLab|LL429 Orcokinin [Asn13]-OrcoKinin NFDEIDRSGFGFN Hoa.SG/CNS
Cmaen PO
NFDEIDRSGFGFN
>gi|430 |LiLab|LL430 Orcokinin [Ser9]-Orcokinin NFDEIDRSSFGFN Csap
SG,PO,br Cmaen Br/VNC/SG/PO Cb br,SG,STG,CoG
NFDEIDRSSFGFN
>gi|431 |LiLab|LL431 Orcokinin [Ser9-val13]-Orcokinin NFDEIDRSSFGFV Csap
SG,PO,br Cmaen VNC/SG/PO Cb br,PO,SG,STG,CoG
NFDEIDRSSFGFV
>gi|432 |LiLab|LL432 Orcokinin [Thr8-His13]-Orcokinin NFDEIDRTGFGFH Csap
br Cb br,PO,SG,STG,CoG
NFDEIDRTGFGFH
>gi|433 |LiLab|LL433 Orcokinin [val13]-OrcoKinin NFDEIDRSGFGFV Csap
SG,PO, br Cmaen VNC/SG/PO Cb br,PO,SG,STG,CoG
NFDEIDRSGFGFV
>gi|434 |LiLab|LL434 Orcokinin Hoa-Orcokinin SSEDMDRLGFG Hoa. SG/CNS
SSEDMDRLGFG
>gi|435 |LiLab|LL435 Orcokinin Hoa-Orcokinin SSEDMPSSLGFGFN Hoa. SG/CNS
SSEDMPSSLGFGFN
>gi|436 |LiLab|LL436 Orcokinin Orcokinin[1-11] NFDEIDRSGFG Csap SG,PO,br
Cmaen Br/VNC Cb br,PO,SGSTG,CoG
NFDEIDRSGFG
>gi|437 |LiLab|LL437 Orcokinin Orcokinin[1-12] NFDEIDRSGFGF Csap SG,PO,br
Cmaen VNC/PO Cb br,PO,SG,STG,CoG
NFDEIDRSGFGF
409
>gi|438 |LiLab|LL438 Orcokinin Orcokinin-like peptides NFDEIDRSSFA Csap
SG,PO,br Cmaen Br
NFDEIDRSSFA
>gi|439 |LiLab|LL439 Orcokinin/Orcomyotropin-related TPRDIANLYamide Csap
SG
TPRDIANLY
>gi|440 |LiLab|LL440 Orcomyotropin FDAFTTGFamide Hoa.CNS
FDAFTTGF
>gi|441 |LiLab|LL441 Orcomyotropin FDAFTTGFGHN Hoa. SG/CNS
FDAFTTGFGHN
>gi|442 |LiLab|LL442 Orcomyotropin
FDAFTTGFGHS Csap PO,SG,br Cmaen
Br/VNC/SG/PO Cb br,PO,SG,STG,CoG
FDAFTTGFGHS
>gi|443 |LiLab|LL443 Orcomyotropin FPAFTTGFGSH Csap SG
FPAFTTGFGSH
>gi|444 |LiLab|LL444 Others AVLLPKKTEKK
AVLLPKKTEKK
>gi|445 |LiLab|LL445 Others DLPKVDTALK
DLPKVDTALK
>gi|446 |LiLab|LL446 Others EVEEPEAPAPPAK
EVEEPEAPAPPAK
>gi|447 |LiLab|LL447 Others GPSGGFNGALAR
GPSGGFNGALAR
>gi|448 |LiLab|LL448 Others
HL/IGSL/IYRamide Csap PO,br Cmaen Br/SG/PO
Cb br,PO,SG,STG,CoG
HLGSLYR
>gi|449 |LiLab|LL449 Others KPKTEKK
KPKTEKK
>gi|450 |LiLab|LL450 Others [Leu]-enkephalin YGGFL
YGGFL
>gi|451 |LiLab|LL451 Others [Met]-enkephalin YGGFM
YGGFM
>gi|452
|LiLab|LL452
PDH(Pigment-dispersing
hormone)
NSELINSLLGISRLMNEAamide Cp.SG
NSELINSLLGISRLMNEA
>gi|453 |LiLab|LL453 PDH(Pigment-dispersing hormone) pQELHVPEREA Csap SG
QELHVPEREA
>gi|454 |LiLab|LL454 PDH(Pigment-dispersing hormone) Pandalus borealis
PDH NSGMINSILGIPRVMTEAamide
NSGMINSILGIPRVMTEA
>gi|455 |LiLab|LL455 PDH(Pigment-dispersing hormone) PDH-armadillidium
QDLNPTEKEVLSNMLDFLQRHSRTTYMFPLLSESKRNSELINSLLGAPRVLNNAamide
armadillidium, predicted
QDLNPTEKEVLSNMLDFLQRHSRTTYMFPLLSESKRNSELINSLLGAPRVLNNA
>gi|456 |LiLab|LL456 PDH(Pigment-dispersing hormone) PDH-Callinectes-I
QELKYQEREMVAELAQQIYRVAQAPWAAAVGPHKRNSELINSILGLPKVMNDAamide Callinectes-I,
predicted
QELKYQEREMVAELAQQIYRVAQAPWAAAVGPHKRNSELINSILGLPKVMNDA
>gi|457 |LiLab|LL457 PDH(Pigment-dispersing hormone) PDH-Callinectes-II
QELHVPEREAVANLAARILKIVHAPHDAAGVPHKRNSELINSLLGISALMNEAamide
CallinectesII, predicted
QELHVPEREAVANLAARILKIVHAPHDAAGVPHKRNSELINSLLGISALMNEA
410
>gi|458
|LiLab|LL458
PDH(Pigment-dispersing
hormone)
PDH-Cancer-I
QDLKYQAREMVAELAQQIYRVAQAPQAGAVGPHKRNSELINSILGLPKVMNDAamide
Cancer-I,
predicted
QDLKYQAREMVAELAQQIYRVAQAPQAGAVGPHKRNSELINSILGLPKVMNDA
>gi|459
|LiLab|LL459
PDH(Pigment-dispersing
hormone)
PDH-Cancer-Iia
QEVNVSEREAVATLAAHILKVVCAPLEGAGGLPHKRNSELINSILGLPKVMNEAamide
Cancer-IIa,
predicted
QEVNVSEREAVATLAAHILKVVCAPLEGAGGLPHKRNSELINSILGLPKVMNEA
>gi|460
|LiLab|LL460
PDH(Pigment-dispersing
hormone)
PDH-Carcinus
QDLKYQEREMVAELAQQIYRVAQAPWAGAVGPHKRNSELINSILGLPKVMNDAamide
carcinus,
predicted
QDLKYQEREMVAELAQQIYRVAQAPWAGAVGPHKRNSELINSILGLPKVMNDA
>gi|461
|LiLab|LL461
PDH(Pigment-dispersing
hormone)
PDH-eurydice
QSRDFSISEREIVASLAKQLLRVARMGYVPEGDLPRKRNAELINSLLGVPRVMSDAamide
eurydice,
predicted
QSRDFSISEREIVASLAKQLLRVARMGYVPEGDLPRKRNAELINSLLGVPRVMSDA
>gi|462 |LiLab|LL462 PDH(Pigment-dispersing hormone) PDH-Marsupenaeus-I
DSSLKYFEREVVSELAAQILRVAQGPSAFVAGPHKRNSELINSLLGIPKVMTDAamide MarsupenaeusI, predicted
DSSLKYFEREVVSELAAQILRVAQGPSAFVAGPHKRNSELINSLLGIPKVMTDA
>gi|463 |LiLab|LL463 PDH(Pigment-dispersing hormone) PDH-Marsupenaeus-II
QREPTASKCQAATELAIQILQAVKGAHTGVAAGPHKRNSELINSLLGLPKFMIDAamide
Marsupenaeus-II, predicted
QREPTASKCQAATELAIQILQAVKGAHTGVAAGPHKRNSELINSLLGLPKFMIDA
>gi|464 |LiLab|LL464 PDH(Pigment-dispersing hormone) PDH-Marsupenaeus-III
QEDLKYFEREVVSELAAQILRVAQGPSAFVAGPHKRNSELINSLLGIPKVMNDAamide MarsupenaeusIII, predicted
QEDLKYFEREVVSELAAQILRVAQGPSAFVAGPHKRNSELINSLLGIPKVMNDA
>gi|465
|LiLab|LL465
PDH(Pigment-dispersing
hormone)
PDH-orconectes
QELKYPEREVVAELAAQIYRVAQAPWAGAVGPHKRNSELINSILGLPKVMNEAamide
orconectes,
predicted
QELKYPEREVVAELAAQIYRVAQAPWAGAVGPHKRNSELINSILGLPKVMNEA
>gi|466 |LiLab|LL466 PDH(Pigment-dispersing hormone) Penaeus aztecus PDH
NSELINSLLGIPKVMNDAamide
NSELINSLLGIPKVMNDA
>gi|467 |LiLab|LL467 PDH(Pigment-dispersing hormone) Procambarus clarkii
PDH NSELINSILGLPKVMNEAamide
NSELINSILGLPKVMNEA
>gi|468 |LiLab|LL468 PDH(Pigment-dispersing hormone) Uca pugilator,
Cancer magister PDH NSELINSILGLPKVMNDAamide Cmaen SG Cb br, SG
NSELINSILGLPKVMNDA
>gi|469 |LiLab|LL469 Proctolin Proctolin RYLPT Csap PO Cmaen PO Cb
br,PO,SG,STG,CoG
RYLPT
>gi|470 |LiLab|LL470 Pyrokinin FSPRLamide Hoa.brain Cb brain
FSPRL
>gi|471 |LiLab|LL471 Pyrokinin LYFAPRLamide Cmaen Br Cb br
LYFAPRL
>gi|472 |LiLab|LL472 Pyrokinin SGGFAFSPRLamide Cb br,STG,CoG
SGGFAFSPRL
>gi|473 |LiLab|LL473 Pyrokinin TDGFAFSPRLamide Cmaen Br
TDGFAFSPRL
>gi|474 |LiLab|LL474 Pyrokinin TSFAFSPRLamide Cmaen Br
TSFAFSPRL
411
>gi|475 |LiLab|LL475
br,CoG
TNFAFSPRL
>gi|476 |LiLab|LL476
AHKNFLRF
>gi|477 |LiLab|LL477
AHRNFLRF
>gi|478 |LiLab|LL478
APNKNFLRF
>gi|479 |LiLab|LL479
APQGNFLRF
>gi|480 |LiLab|LL480
APRNFLRF
>gi|481 |LiLab|LL481
ARPRNFLRF
>gi|482 |LiLab|LL482
ASNNLRF
>gi|483 |LiLab|LL483
AYPSLRLRF
>gi|484 |LiLab|LL484
DARTAPLRLRF
>gi|485 |LiLab|LL485
DENRNFLRF
>gi|486 |LiLab|LL486
DGNRNFLRF
>gi|487 |LiLab|LL487
DGPLAPFLRF
>gi|488 |LiLab|LL488
DHVPFLRF
>gi|489 |LiLab|LL489
DQNRNFLRF
>gi|490 |LiLab|LL490
DRNFVLRF
>gi|491 |LiLab|LL491
DTSTPALRLRF
>gi|492 |LiLab|LL492
EFLRF
>gi|493 |LiLab|LL493
EFVDPNLRF
>gi|494 |LiLab|LL494
ELNFLRF
>gi|495 |LiLab|LL495
EMPSLRLRF
>gi|496 |LiLab|LL496
ENRNFLRF
>gi|497 |LiLab|LL497
EPLDHVFLRF
>gi|498 |LiLab|LL498
ETNFLRF
>gi|499 |LiLab|LL499
FDDFLRF
>gi|500 |LiLab|LL500
FEPSLRLRF
>gi|501 |LiLab|LL501
Pyrokinin Cb Pyrokinin I TNFAFSPRLamide Csap PO Cb
RFamide
AHKNFLRFamide Csap PO Cb PO
RFamide
AHRNFLRFamide Cb PO
RFamide
APNKNFLRFamide Cb PO
RFamide
APQGNFLRFamide Csap PO Cmaen Br/PO
RFamide
APRNFLRFamide Cmaen VNC/PO Cb br CoG
RFamide
ARPRNFLRFamide Cb PO
RFamide
ASNNLRFamide Csap PO
RFamide
AYPSLRLRFamide Hoa.CNS
RFamide
DARTAPLRLRFamide Csap br Cmaen Br/VNC
RFamide
DENRNFLRFamide Csap PO, Cb PO
RFamide
DGNRNFLRFamide Cmaen Br/VNC/PO
RFamide
DGPLAPFLRFamide Csap PO
RFamide
DHVPFLRFamide Cb PO
RFamide
DQNRNFLRFamide Hoa.CNS
RFamide
DRNFVLRFamide Cb PO
RFamide
DTSTPALRLRFamide Hoa.CNS
RFamide
EFLRFamide Csap PO
RFamide
EFVDPNLRFamide Csap PO
RFamide
ELNFLRFamide Csap PO
RFamide
EMPSLRLRFamide Cmaen VNC
RFamide
ENRNFLRFamide Cb PO
RFamide
EPLDHVFLRFamide Csap
RFamide
ETNFLRFamide Csap PO
RFamide
FDDFLRFamide Csap PO
RFamide
FEPSLRLRFamide Hoa.CNS
RFamide
FTSKNYLRFamide Cb,PO,STG
412
FTSKNYLRF
>gi|502 |LiLab|LL502 RFamide
GAHKNYLRFamide Csap SG,PO,br Cmaen PO Cb
br,PO,STG,CoG
GAHKNYLRF
>gi|503 |LiLab|LL503 RFamide GGGEYDDYGHLRFamide Hoa.CNS
GGGEYDDYGHLRF
>gi|504 |LiLab|LL504 RFamide GHRNFLRFamide Cb PO
GHRNFLRF
>gi|505 |LiLab|LL505 RFamide GLSRNYLRFamide Csap PO,br Cmaen Br/VNC/PO
GLSRNYLRF
>gi|506 |LiLab|LL506 RFamide
GNRNFLRFamide Csap PO Cmaen Br/PO Cb
br,PO,STG,CoG
GNRNFLRF
>gi|507 |LiLab|LL507 RFamide GPFLRFamide Csap PO Cb PO
GPFLRF
>gi|508 |LiLab|LL508 RFamide GPKNFLRFamide Csap
GPKNFLRF
>gi|509 |LiLab|LL509 RFamide GPPSLRLRFamide Hoa.CNS
GPPSLRLRF
>gi|510 |LiLab|LL510 RFamide GPRNFLRFamide Csap PO
GPRNFLRF
>gi|511 |LiLab|LL511 RFamide GRNFLRFamide Cb br,PO
GRNFLRF
>gi|512 |LiLab|LL512 RFamide GYNRSFLRFamide
GYNRSFLRF
>gi|513 |LiLab|LL513 RFamide GYSDRNYLRFamide Hoa.CNS
GYSDRNYLRF
>gi|514 |LiLab|LL514 RFamide GYSKNYLRFamide Csap PO Cb br,PO,STG,CoG
GYSKNYLRF
>gi|515 |LiLab|LL515 RFamide GYSVGLNYLRFamide Csap PO
GYSVGLNYLRF
>gi|516 |LiLab|LL516 RFamide HDLVQVFLRFamide Csap
HDLVQVFLRF
>gi|517 |LiLab|LL517 RFamide HDSPHVFLRFamide Csap
HDSPHVFLRF
>gi|518 |LiLab|LL518 RFamide HPLSFVSALRFamide Csap
HPLSFVSALRF
>gi|519 |LiLab|LL519 RFamide HVFLRFamide Csap PO
HVFLRF
>gi|520 |LiLab|LL520 RFamide LAPQRNFLRFamide Csap
LAPQRNFLRF
>gi|521 |LiLab|LL521 RFamide LAYNRSFLRFamide Csap
LAYNRSFLRF
>gi|522 |LiLab|LL522 RFamide LDGPLAPFLRFamide Csap
LDGPLAPFLRF
>gi|523 |LiLab|LL523 RFamide LDRNFLRFamide Csap
LDRNFLRF
>gi|524 |LiLab|LL524 RFamide LELNFLRFamide Csap
LELNFLRF
>gi|525 |LiLab|LL525 RFamide LETNFLRFamide Csap
LETNFLRF
>gi|526 |LiLab|LL526 RFamide LFDDFLRFamide Csap
LFDDFLRF
>gi|527 |LiLab|LL527 RFamide LGDRNFLRFamide Csap
413
LGDRNFLRF
>gi|528 |LiLab|LL528
LGRPNFLRF
>gi|529 |LiLab|LL529
LNPFLRF
>gi|530 |LiLab|LL530
LNPSNFLRF
>gi|531 |LiLab|LL531
LNQPNFLRF
>gi|532 |LiLab|LL532
LNRNFLRF
>gi|533 |LiLab|LL533
LNRSFLRF
>gi|534 |LiLab|LL534
LPGVNFLRF
>gi|535 |LiLab|LL535
LRNLRF
>gi|536 |LiLab|LL536
LRQFLRF
>gi|537 |LiLab|LL537
LSPRNFLRF
>gi|538 |LiLab|LL538
LTGNRNFLRF
>gi|539 |LiLab|LL539
LTHPFLRF
>gi|540 |LiLab|LL540
LTNRNFLRF
>gi|541 |LiLab|LL541
LTNYGGFLRF
>gi|542 |LiLab|LL542
LTRPLRF
>gi|543 |LiLab|LL543
LYEQDFLRF
>gi|544 |LiLab|LL544
MPYLRF
>gi|545 |LiLab|LL545
NFLRF
>gi|546 |LiLab|LL546
NPSDFLRF
>gi|547 |LiLab|LL547
NPSNFLRF
>gi|548 |LiLab|LL548
NQPNFLRF
>gi|549 |LiLab|LL549
br,PO
NRSFLRF
>gi|550 |LiLab|LL550
PGVNFLRF
>gi|551 |LiLab|LL551
PKSNFLRF
>gi|552 |LiLab|LL552
QDNDHVFLRF
>gi|553 |LiLab|LL553
QGNFLRF
RFamide
LGRPNFLRFamide Csap
RFamide
LNPFLRFamide
RFamide
LNPSNFLRFamide Csap
RFamide
LNQPNFLRFamide Csap
RFamide
LNRNFLRFamide Csap
RFamide
LNRSFLRFamide Csap
RFamide
LPGVNFLRFamide Csap
RFamide
LRNLRFamide Csap PO
RFamide
LRQFLRFamide Csap
RFamide
LSPRNFLRFamide Csap
RFamide
LTGNRNFLRFamide Csap
RFamide
LTHPFLRFamide Csap
RFamide
LTNRNFLRFamide Csap
RFamide
LTNYGGFLRFamide Csap
RFamide
LTRPLRFamide Csap br
RFamide
LYEQDFLRFamide Csap
RFamide
MPYLRFamide Csap PO
RFamide
NFLRFamide Hoa.CNS Csap PO Cb br PO
RFamide
NPSDFLRFamide Csap PO Cb PO
RFamide
NPSNFLRFamide Csap PO Cb PO
RFamide
NQPNFLRFamide Csap PO
RFamide
NRSFLRFamide Csap PO,br Cmaen Br/VNC/PO Cb
RFamide
PGVNFLRFamide Csap PO
RFamide
PKSNFLRFamide Cb PO
RFamide
pQDNDHVFLRFamide Csap br
RFamide
pQGNFLRFamide Csap PO Cmaen PO
414
>gi|554 |LiLab|LL554 RFamide pQRNFLRFamide Cb br,PO
QRNFLRF
>gi|555 |LiLab|LL555 RFamide PSLRLRFamide Hoa.CNS Cmaen Br/VNC Cb br
PSLRLRF
>gi|556 |LiLab|LL556 RFamide PSMRLRFamide Csap br Cmaen Br/VNC Cb br CoG
PSMRLRF
>gi|557 |LiLab|LL557 RFamide QYFMRLFRamide Csap PO
QYFMRLFR
>gi|558 |LiLab|LL558 RFamide RDRNFLRFamide Cb br,PO
RDRNFLRF
>gi|559 |LiLab|LL559 RFamide RNFLRFamide Csap PO Cmaen Br/VNC/PO Cb br
PO
RNFLRF
>gi|560 |LiLab|LL560 RFamide RQFLRFamide Csap
RQFLRF
>gi|561 |LiLab|LL561 RFamide RSFLRFamide Cp.PO
RSFLRF
>gi|562 |LiLab|LL562 RFamide SDRNYLRFamide
SDRNYLRF
>gi|563 |LiLab|LL563 RFamide
SENRNFLRFamide Csap PO Cmaen VNC Cb
br,PO,STG,CoG
SENRNFLRF
>gi|564 |LiLab|LL564 RFamide SGRNFLRFamide Hoa.CNS
SGRNFLRF
>gi|565 |LiLab|LL565 RFamide SKNYLRFamide Csap PO Cb PO CoG
SKNYLRF
>gi|566 |LiLab|LL566 RFamide SMPTLRLRFamide Csap br
SMPTLRLRF
>gi|567 |LiLab|LL567 RFamide SPRNFLRFamide Csap PO
SPRNFLRF
>gi|568 |LiLab|LL568 RFamide SQGLNSDLRFamide Csap PO
SQGLNSDLRF
>gi|569 |LiLab|LL569 RFamide SQPSKNYLRFamide Csap PO Cb PO
SQPSKNYLRF
>gi|570 |LiLab|LL570 RFamide SRNYLRFamide Cmaen Br/VNC/PO
SRNYLRF
>gi|571 |LiLab|LL571 RFamide TGNRNFLRFamide Csap PO
TGNRNFLRF
>gi|572 |LiLab|LL572 RFamide THPFLRFamide Csap PO
THPFLRF
>gi|573 |LiLab|LL573 RFamide TNYGGFLRFamide Csap PO
TNYGGFLRF
>gi|574 |LiLab|LL574 RFamide TRNFLRFamide
TRNFLRF
>gi|575 |LiLab|LL575 RFamide YEQDFLRFamide Csap PO
YEQDFLRF
>gi|576 |LiLab|LL576 RFamide YGNRPHLRFamide Csap
YGNRPHLRF
>gi|577 |LiLab|LL577 RFamide YGSDRNFLRFamide Cb PO
YGSDRNFLRF
>gi|578 |LiLab|LL578 RFamide [ala1]-FaRP AYNRSFLRFamide Csap PO Cb
br,PO,STG,CoG
AYNRSFLRF
415
>gi|579 |LiLab|LL579 RFamide [glu2-leu3]-SchistoFLRFa pQDLDHVFLRFamide
Csap PO,br Cmaen Br/VNC/PO Cb br,PO,STG,CoG
QDLDHVFLRF
>gi|580 |LiLab|LL580 RFamide [val2] pem-FLP 6 DVRTPALRLRFamide Cb br,CoG
DVRTPALRLRF
>gi|581 |LiLab|LL581 RFamide DF 2, Mar-FLP 1, PrcFaRP 5 DRNFLRFamide Csap
PO,br Cmaen Br/SG/PO Cb br,PO,STG,CoG
DRNFLRF
>gi|582 |LiLab|LL582 RFamide F1 SDRNFLRFamide
SDRNFLRF
>gi|583 |LiLab|LL583 RFamide F2 TNRNFLRFamide Csap PO
TNRNFLRF
>gi|584
|LiLab|LL584
RFamide
Lepidopteran
peptide
FLRFamide
I
pEDVVHSFLRFamide
QDVVHSFLRF
>gi|585
|LiLab|LL585
RFamide
Lepidopteran
peptide
FLRFamide
II
GNSFLRFamide
GNSFLRF
>gi|586
|LiLab|LL586
RFamide
Lepidopteran
peptide
FLRFamide
III
DPSFLEFamide
DPSFLEF
>gi|587 |LiLab|LL587 RFamide Mar-FLP 2 ADKNFLRFamide
ADKNFLRF
>gi|588 |LiLab|LL588 RFamide Mar-FLP 3 NYDKNFLRFamide
NYDKNFLRF
>gi|589 |LiLab|LL589 RFamide Mar-FLP 4, sNPF (short neuropeptide F)
APALRLRFamide
APALRLRF
>gi|590 |LiLab|LL590 RFamide Mar-FLP 5 DRTPALRLRFamide
DRTPALRLRF
>gi|591 |LiLab|LL591 RFamide Mar-FLP 6 DGGRNFLRFamide
DGGRNFLRF
>gi|592 |LiLab|LL592 RFamide Mar-FLP 7 GYGDRNFLRFamide
GYGDRNFLRF
>gi|593 |LiLab|LL593 RFamide Mar-FLP 8 VSHNNFLRFamide
VSHNNFLRF
>gi|594 |LiLab|LL594 RFamide NF 1, PrcFaRP 2 NRNFLRFamide Csap PO,br
Cmaen Br/VNC/SG/PO Cb br,PO,STG,CoG
NRNFLRF
>gi|595
|LiLab|LL595
RFamide
NPF
(neuropeptide
F)
KPDPSQLANMAEALKYLEQELDKYYSQVSRPRFamide
KPDPSQLANMAEALKYLEQELDKYYSQVSRPRF
>gi|596 |LiLab|LL596 RFamide NPY/PP peptide pem-PYF 3 YAIAGRPRFamide
YAIAGRPRF
>gi|597 |LiLab|LL597 RFamide NPY/PP peptide pem-PYF2 YSQVSRPRFamide
YSQVSRPRF
>gi|598 |LiLab|LL598 RFamide NPY/PP peptide pem-PYF4 YSLRARPRFamide
YSLRARPRF
>gi|599 |LiLab|LL599 RFamide pem-FLP 1 GDRNFLRFamide
GDRNFLRF
>gi|600 |LiLab|LL600 RFamide pem-FLP 2 AYSNLNYLRFamide
AYSNLNYLRF
>gi|601 |LiLab|LL601 RFamide pem-FLP 3 AQPSMRLRFamide
AQPSMRLRF
416
>gi|602 |LiLab|LL602 RFamide pem-FLP 4 SQPSMRLRFamide
SQPSMRLRF
>gi|603 |LiLab|LL603 RFamide pem-FLP 5 SMPSLRLRFamide Cmaen Br/VNC/SG Cb
br,CoG
SMPSLRLRF
>gi|604 |LiLab|LL604 RFamide pem-FLP 6 DGRTPALRLRFamide
DGRTPALRLRF
>gi|605 |LiLab|LL605 RFamide pem-PYF1 RARPRFamide
RARPRF
>gi|606 |LiLab|LL606 RFamide PrcFaRP 1 FLRFamide AYSDRNFLRFamide
AYSDRNFLRF
>gi|607 |LiLab|LL607 RFamide PrcFaRP 1 YLRFamide AYSDRNYLRFamide
AYSDRNYLRF
>gi|608 |LiLab|LL608 RFamide PrcFaRP 3 SRNFLRFamide
SRNFLRF
>gi|609 |LiLab|LL609 RFamide PrcFaRP 4 ALDRNFLRFamide
ALDRNFLRF
>gi|610 |LiLab|LL610 RFamide PrcFaRP 6 APQRNFLRFamide Csap PO,br Cmaen
VNC Cb br,PO,STG,CoG
APQRNFLRF
>gi|611 |LiLab|LL611 RFamide S I/LNFTHKFamide
I/LNFTHKF
>gi|612 |LiLab|LL612 RFamide sulfakinin pQFDEY(sulf)GHMRFamide
QFDEYGHMRF
>gi|613 |LiLab|LL613 RPCH Anaim-AKH pEVNFSPSWamide Anax imperator
EVNFSPSW
>gi|614 |LiLab|LL614 RPCH Corpu-AKH pELNFSPSWamide Corixa punctata
ELNFSPSW
>gi|615 |LiLab|LL615 RPCH Dappu-RPCH pEVNFSTSWamide Daphnia pulex,
predicted
EVNFSTSW
>gi|616 |LiLab|LL616 RPCH Grybi-Akh pEVNFSTGWamide Gryllus bimaculatus
EVNFSTGW
>gi|617 |LiLab|LL617 RPCH Ile2-Panbo-RPCH pEINFSPGWamide unnatural
EINFSPGW
>gi|618 |LiLab|LL618 RPCH Manto-CC pEVNFSPGWamide species of order
Mantophasmatodea
EVNFSPGW
>gi|619 |LiLab|LL619 RPCH Nepci-AKH pELNFSSGWamide Nepa cinerea
ELNFSSGW
>gi|620 |LiLab|LL620 RPCH Panbo-RPCH pELNFSPGWamide Pandalus borealis
ELNFSPGW
>gi|621 |LiLab|LL621 RPCH RPCH pQLNFSPGWamide Csap SG,br Cmaen SG Cb SG
QLNFSPGW
>gi|622 |LiLab|LL622 RPCH Schgr-AKH-III pELNFSTGWamide Schistocera
gregaria
ELNFSTGW
>gi|623 |LiLab|LL623 RYamide (X)YANRYamide Cmaen PO
YANRY
>gi|624 |LiLab|LL624 RYamide EWYSQRYamide Csap PO
EWYSQRY
>gi|625 |LiLab|LL625 RYamide FVGGSRYamide Csap PO Cmaen PO Cb br,PO,SG
FVGGSRY
>gi|626 |LiLab|LL626 RYamide FVNSRYamide
417
FVNSRY
>gi|627 |LiLab|LL627 RYamide FYANRYamide Csap PO Cmaen PO Cb PO
FYANRY
>gi|628 |LiLab|LL628 RYamide FYSQRYamide Csap PO Cmaen PO Cb PO
FYSQRY
>gi|629 |LiLab|LL629 RYamide GFVSNRYamide Csap PO
GFVSNRY
>gi|630 |LiLab|LL630 RYamide L/IFVGGSRYamide Cp.PO
LFVGGSRY
>gi|631 |LiLab|LL631 RYamide LEWYSQRYamide Csap
LEWYSQRY
>gi|632 |LiLab|LL632 RYamide LFYSQRYamide Csap
LFYSQRY
>gi|633 |LiLab|LL633 RYamide LGFVSNRYamide Csap
LGFVSNRY
>gi|634 |LiLab|LL634 RYamide LSGFYANRYamide Csap
LSGFYANRY
>gi|635 |LiLab|LL635 RYamide LSSRFVGGSRYamide Csap
LSSRFVGGSRY
>gi|636 |LiLab|LL636 RYamide PAFYSQRYamide
PAFYSQRY
>gi|637 |LiLab|LL637 RYamide
pQGFYSQRYamide Csap SG,PO Cmaen PO Cb
br,PO,SG,STG,CoG
QGFYSQRY
>gi|638 |LiLab|LL638 RYamide RFVGGSRYamide Cp.PO
RFVGGSRY
>gi|639 |LiLab|LL639 RYamide RFYANRYamide Csap PO
RFYANRY
>gi|640 |LiLab|LL640 RYamide SGFYADRYamide Cmaen PO
SGFYADRY
>gi|641 |LiLab|LL641 RYamide SGFYANRYamide Csap PO Cmaen PO Cb PO
SGFYANRY
>gi|642 |LiLab|LL642 RYamide SGFYAPRYamide Csap PO Cmaen PO
SGFYAPRY
>gi|643 |LiLab|LL643 RYamide SRFVGGSRYamide Csap PO
SRFVGGSRY
>gi|644 |LiLab|LL644 RYamide SSRFVGGSRYamide Csap PO Cmaen PO Cb PO
SSRFVGGSRY
>gi|645 |LiLab|LL645 RYamide VGFYANRYamide Csap PO
VGFYANRY
>gi|646 |LiLab|LL646 SIFamide AYRKPPFNGSIFamide Insect
AYRKPPFNGSIF
>gi|647 |LiLab|LL647 SIFamide
RKPPFNGSIFamide Hoa.CNS Cmaen Br
Cb
br,STG,CoG
RKPPFNGSIF
>gi|648 |LiLab|LL648 SIFamide VYRKPPFNGSIFamide Hoa.CNS Cmaen VNC
VYRKPPFNGSIF
>gi|649 |LiLab|LL649 SIFamide pem-FLP 7 GYRKPPFNGSIFamide Csap br Cmaen
Cmaen br/VCN/SG Cb br,SG,STG,CoG
GYRKPPFNGSIF
>gi|650 |LiLab|LL650 Tachykinin APSGFLGM Csap br
APSGFLGM
>gi|651 |LiLab|LL651 Tachykinin APSGFLGMRamide-NH3-loss Csap br
APSGFLGMR
418
>gi|652 |LiLab|LL652
APSGFLGMRG
>gi|653 |LiLab|LL653
PSGFLGMR
>gi|654 |LiLab|LL654
Br/VNC/SG Cb br,CoG
TPSGFLGMR
>gi|655 |LiLab|LL655
br,STG,CoG
SGFLGMR
>gi|656 |LiLab|LL656
YPSGFLGMR
>gi|657 |LiLab|LL657
APSFGQ
>gi|658 |LiLab|LL658
GFLGMR
Tachykinin
APSGFLGMRG Csap PO Cmaen Br Cb SG,CoG
Tachykinin
PSGFLGMRamide Cmaen Br/VNC/SG
Tachykinin CabTRP II TPSGFLGMRamide Csap PO Cmaen
Tachykinin CabTRP lb SGFLGMRamide Cmaen Br/VNC Cb
Tachykinin CalsTRP YPSGFLGMRamide Csap br
Tachykinin TRP APSFGQamide
Tachykinin TRP GFLGMRamide
Neuropeptides Found in Orconectes rusticus
Neuropeptide Family NP Matching Sequence
AST-B
TNWNKFQGSWamide
FMRFamide
EPLDHVFLRFamide
FMRFamide
EPLDHVFLRFamide
FMRFamide
EPLDHVFLRFamide
FMRFamide
YAIAGRPRFamide
Myosuppressin
pQDLDHVFLRFamide
Myosuppressin
pQDLDHVFLRFamide
Myosuppressin
pQDLDHVFLRFamide
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcokinin
Orcomyotropin
FDAFTTGFGHS
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
Sequence of PSM
TNWNKFQGSWa
EPLDHVFLR
EPLDHVFLRF
EPLDHVFLRFa
YAIAGRPR
pQDLDHVFLR
pQDLDHVFLRF
pQDLDHVFLRFa
DFDEIDRSGFA
EIDRSGFGFN
FDEIDRSGFGFN
NFDEIDR
NFDEIDRS
NFDEIDRSGFA
NFDEIDRSGFG
NFDEIDRSGFGF
NFDEIDRSGFGFA
NFDEIDRSGFGFN
NFDEIDRSGFGFV
NFDEIDRSSFG
NFDEIDRTGFa
NFDEIDRTGFG
NFDEIDRTGFGF
NFDEIDRTGFGFH
FDAFTTGFGHS
GSSRMERLL
LLSSGSSSSEPLS
LLSSGSSSSEPLSF
LLSSGSSSSEPLSFL
LLSSGSSSSEPLSFLSQ
LLSSGSSSSEPLSFLSQDQSVN
LLSSGSSSSEPLSFLSQDQSVS
LSSGSSSSEPLSFL
LSSGSSSSEPLSFLSQDQSVS
MERLLSSGSSSSEPLSFLSQDQSVS
PLSFLSQDQSVS
RLLSSGSSSSEPL
RLLSSGSSSSEPLS
RLLSSGSSSSEPLSF
RLLSSGSSSSEPLSFL
RLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRM(O)ERLLSSGSSSSEPLSFLSQDQSVS
RSVEGSSRMERL
RSVEGSSRMERLL
25.83
43.97
64.89
51.49
83.14
109.36
21.4
45.31
116.9
64.49
40.65
63.06
70.99
54.37
92.46
64.5
37.43
21.59
53.36
46.01
75.71
49.52
19.91
18.8
33.24
29.98
53.35
Eyestalk
PEAKS Score (-10logP)
32.36
Appendix C. Neuropeptides and Proteins Identified in Orconectes rusticus
33.81
151.69
82.22
46.53
94.94
92.77
59.16
100.88
47.02
28.97
15.38
63.15
16.43
33.61
24.56
34.29
58.65
51.41
110.22
28.28
34.47
64.87
41.7
22.13
35.44
16.55
48.09
35.44
16.55
51.99
Mascot Score
27.52
38.98
51.82
58.31
75.5
48.84
76.44
69.98
16.85
23.52
29.92
45.87
30.71
32.87
Brain
PEAKS Score (-10logP)
21.27
43.02
36.02
46.55
45.69
101.5
58.89
46.25
33.66
47.52
20.73
Mascot Score
419
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
CPRP
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
PDH
SIFamide
SIFamide
SIFamide
SIFamide
SIFamide
SIFamide
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVN
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVNa
RSVEGSSRMERLLSSGSSSSEPLSFLSQDQSVS
SEPLSFLSQDQSVS
SGSSSSEPLSFL
SGSSSSEPLSFLSQDQSVN
SGSSSSEPLSFLSQDQSVS
SSEPLSFLSQDQSVS
SSGSSSSEPLSFLSQDHSV
SSGSSSSEPLSFLSQDQSVS
SSSEPLSFLSQDQSVS
SSSSEPLSFLSQDQSVS
AAQllRVAQGPSAFVAGPHK
AGGlPHKRNa
AQGPSAFVAGPHK
DlNPTEKa
EAVATlAAHllKVVCAPlEGAGGlPHKa
EYllKFa
lGTM(O)AGGPHKa
llGlPKVM(O)T
llGlPKVMNEAa
MVAM(O)SlQlT
NSEllNAllGSPTl
NSEllNAllGSPTlM
NSEllNSllGl
NSEllNSllGlP
NSEllNSllGlPK
NSEllNSllGlPKVMNEAa
pQElKYPEREVVAEl
pQElKYPEREVVAElA
pQElKYPEREVVAElAA
pQElKYPEREVVAElAAQ
SEllNSllGlPa
SllGlPKVM(O)
SllGlPKVMNEAa
SlM(O)SEEVa
SlTM(O)DVES
SSlKYFEREVVSElAAQll
SSlKYFEREVVSElAAQllR
SSlKYFEREVVSElAAQllRV
VAQGPSAFVAGPHK
VVlAAVlTQG
GYRKPPFN
GYRKPPFNG
GYRKPPFNGSIF
GYRKPPFNGSIFa
KPPFNGSIF
KPPFNGSIFa
19.79
24.71
18.82
30.95
15.39
24.73
33.91
19.25
30.71
56.9
64.97
21.04
20.16
28.04
34.24
49.1
53.8
36.15
17.94
19.05
19.96
25.29
31.32
25.71
38.13
41.45
65.89
76.48
102.29
19.06
45.91
21.47
32.27
50.76
82.63
67
16.23
16.66
26.61
29.27
48.58
73.48
20.08
36.65
69.16
38.04
21.71
24.69
87.6
28.58
34.91
86.67
35.58
38.6
81.38
71.45
129.83
16.35
15.74
19.11
47.45
21.08
55.2
51.55
17
21.09
16.58
29.83
19.03
36.46
37.37
16.36
25.71
420
SIFamide
SIFamide
SIFamide
SIFamide
SIFamide
SIFamide
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
GYRKPPFNGSIFa
RKPPF
RKPPFN
RKPPFNG
RKPPFNGSI
RKPPFNGSIF
RKPPFNGSIFa
30.91
39.46
38.42
27.45
21.18
23.42
23.7
33.66
21.41
17.23
421
CHH
CHH
CPRP
CPRP
CPRP
CPRP
Crustacean female
hormone
Crustacean female
hormone
Crustacean female
hormone
EH
Eyestalk peptide
Eyestalk peptide
FMRFamide
FMRFamide
Insulin-like
O15981.1
O97384.1
AAM21927.1
AAS46643.1
ABS01332.1
LL343
ADO00266.1
AFK81936.1
AF112986_1
AF112986_1
BAE06263.1
EFX80896.1
ADA67878.1
ADO00266.1
ADO00266.1
CHH
LL206
NPs Found in O. rusticus, novel
Accession
Number
Peptide Family
ABX55995.1
Bursicon
ACG50067.1
Bursicon
ACG50067.1
Bursicon
ACG50067.1
Bursicon
ADI86242.1
Bursicon
ACX46386.1
Calcitonin
ACX46386.1
Calcitonin
ABB46292.1
CCAP
BAF34909.1
CCAP
BAF34909.1
CCAP
AAD45236.1
CHH
ACN65120.1
CHH
ACS35346.1
CHH
AER27833.1
CHH
AFD28272.1
CHH
AFD28272.1
CHH
AFG16934.1
CHH
AFM29133.1
CHH
crustacean female hormone [Callinectes sapidus]
eclosion hormone [Amphibalanus amphitrite]
eyestalk peptide [Jasus edwardsii]
eyestalk peptide [Jasus edwardsii]
FLRFamide precursor protein B [Procambarus clarkii]
putative sulfakinin-like peptide [Daphnia pulex]
insulin-like androgenic gland hormone precursor [Penaeus monodon]
crustacean female hormone [Callinectes sapidus]
crustacean female hormone [Callinectes sapidus]
Description
bursicon alpha [Carcinus maenas]
bursicon hormone alpha subunit [Callinectes sapidus]
bursicon hormone alpha subunit [Callinectes sapidus]
bursicon hormone alpha subunit [Callinectes sapidus]
bursicon alpha subunit [Homarus gammarus]
prepro-calcitonin-like diuretic hormone [Homarus americanus]
prepro-calcitonin-like diuretic hormone [Homarus americanus]
crustacean cardioactive peptide [Homarus gammarus]
crustacean cardioactive peptide [Procambarus clarkii]
crustacean cardioactive peptide [Procambarus clarkii]
hyperglycemic hormone-like neuropeptide 43-4 [Metapenaeus ensis]
crustacean hyperglycemic hormone [Panulirus homarus]
Crustacean hyperglycemic hormone isoform 1 [Rimicaris kairei]
Crustacean hyperglycaemic hormone [Ptychognathus pusillus]
CHH protein [Scylla paramamosain]
CHH protein [Scylla paramamosain]
Hyperglycemic hormone [Pandalopsis japonica]
Crustacean hyperglycmic hormone [Portunus pelagicus]
Crustacean hyperglycemic hormone Hoa-CHH-A (pCHH-A[pQ61V132amide]) [Homarus amerianus]
Crustacean hyperglycemic hormones 5; Pej-SGP-V [Marsupenaeus
japonicus]
Crustacean hyperglycemic hormone 2; Pm-SGP-II [Penaeus
monodon]
Crustacean hyperglycemic hormone precursor [Pachygrapsus
marmoratus]
cCustacean hyperglycemic hormone precursor [Macrobrachium
rosenbergii]
Prepro-crustacean hyperglycemic hormone [Galathea strigosa]
CHH precursor-related peptide Capr CPRP II [13-38] [Cancer
productus]
QGPLPIQPVa
PGLASLGGSTST
DVRQDGQTNa
TPARQR
PAEYSRNVR
PVELGSSNPa
VGMLMVLSLT
ERQIQGP
DGGYIDLSENa
GDLPGGLVHamide
RIEKLLST
LSFMPEHP
DRSLFGKL
SSPVASLIRG
LVAGASSAGT
LDDLLLSDV
PSM Sequence
VTVLVVIG
AM(O)VGAAVT
M(O)VGAAVTV
VLTRAPIDCMCRPCT
TVVWSDECSLT
LTRLGHSIIRANELEKFVRSSGSA
TRLGHSIIRANELEKF
TPHTQPR
AGPLAKRDIG
KLWEQLQ
pEAASPGASSPWVEHR
SPPPAGRTATVIGCSVNVSTTYa
HPSGLAALTASH
PAGHPLEKRQamide
LVACIAMATLPQTQamide
pEGAAHPLEKRamide
EEHVDIR
APM(O)QGYGTEamide
20.84
20.02
17.36
15.07
21.73
15.63
15.04
21.37
19.2
22.2
15.02
17.91
PEAKS
Eyestalk
30.15
28.32
19.46
Mascot
37.72
19.38
19.84
15.44
17.93
18.55
16.77
18.19
19.29
18.05
16.15
22.81
21.51
15.38
18.99
PEAKS
Brain
18.3
18.17
28.86
58.81
Mascot
422
Orcokinin
Orcokinin
Orcokinin
Oxytocinneurophysin
RPCH
RPCH
SIFamide-related
peptide
Tachykinin
Tachykinin
Tachykinin
Q9NL83.1
Q9NL83.1
Q9NL83.2
Q867W1.1
BAC82426.1
BAC82426.1
BAC82426.1
ADD24046.1
ADN95181.1
ADQ73633.1
Insulin-like
Insulin-like
Insulin-like
EFX70023.1
EFX76240.1
EFX76240.1
GYRKPPFNGSIF-amide; FRP1_PROCL [Procambarus clarkii]
preprotachykinin [Procambarus clarkii]
preprotachykinin [Procambarus clarkii]
preprotachykinin [Procambarus clarkii]
Oxytocin-neurophysin 1 [Lepeophtheirus salmonis]
red pigment concentrating hormone precursor [Scylla paramamosain]
red pigment concentrating hormone precursor [Scylla olivacea]
Insulin/IGF/relaxin-like peptide 2 [Daphnia pulex]
insulin/IGF/relaxin-like peptide 1 [Daphnia pulex]
insulin/IGF/relaxin-like peptide 1 [Daphnia pulex]
Orcomyotropin/orcokinin precursor-like peptide [Procambarus
clarkii]
Orcomyotropin/orcokinin precursor-like peptide [Procambarus
clarkii]
Orcomyotropin/orcokinin precursor-like peptide [Procambarus
clarkii]
AGGDSLYEPGK
AGMDSELETLL
HFDDESEIDAY
HFDDESEIDAYIQAL
QYKQCSSCGP
SVGGAPGGVVPSSPGSSSGDSa
PPGSSSGDSCGP
VYVPRYIANLY
VYVPRYIAN
MTAQM(O)FTIALLLSLS
SLGLTTTAATPNa
M(O)IIPSTVGRCWM(O)
VTPAEP
51.8
17.89
38.47
77.24
38.23
18.39
21.23
45.72
22.85
18.11
18.54
18.49
45.89
16.8
37.25
87.4
29.9
22.28
22.03
423
EGFAPHTTFGT
E(-18.01)LGDAPDVAAEKAR
EGFAPHTTCST
DPSGSTLAQR
DSYGYHLDR
DSYGYHLDR
E(-18.01)EEVAAVG
DPSGSTLAQR
DGFAPHTTFGT
DLPVSAAPVVESAPVVES
DPSGLELAGA
DPSGLPLAAA
DPSGLPLAAA
DPSGLPLAAA
DPSGLPLAQ
DPSGLPLAQ
DPSGLPLAQ
DPSGPALAKAA
DPSGPALANR
DGAPAAEDVKAPES
DGAPAAEDVKAPES
APVLGYWNLR
ATLGVGAE
AVGFLPALNLT
APVFGYWNLR
ALVGPSGLLLDDGTPVQFK
ADVPDLGA
AGFAGDDANGP(-.98)
ADVPDLAA
AALPLGNKEKTGA
AAPPPVPTPEPT
Peptide
Best BLAST
E-value
XP_001951442 predicted serine/threonine protein
kindase MAKlike Acyrthosiphon pisum
27
EFX85740 hypothetical protein dappudraft_309057
0.046
ACO14744.1 general transcription factor IIF subunit 3
Caligus clemensi
6.1
ACO15766.1 U3 small nucleolar ribonucleoprotein
protein IMP4 Caligus clemensi
6.1
ACT82962.1 beta-actin Penaeus monodon
0.28
ABM54460.1 cuticle protein CUT 9 Portunus
pelagicus
2.00E-08
ADV59553.1
glutathione
S-transferase
mu
Parcyclopina nana
0.069
ADV59553.1
glutathione
S-transferase
mu
Parcyclopina nana
0.069
ACO15187.1 tetraspanin-13 Caligus clemensi
4.4
EFX61408.1 hypothetical protein dappudrapt_339726
2.3
ACO12558.1 tissue factor pathway nhibitor precursor
Lepeophtheirus salmonis
0.86
ACO12558.1 tissue factor pathway nhibitor precursor
Lepeophtheirus salmonis
0.86
ABV58636.1 hemocyanin-like protein Metapenaeus
ensis
0.03
EFX64340.1 hypothetical protein dappudraft_305068
0.006
EFX60846.1 hypothetical protein dappudraft_341180
9.5
EFX78346.1hypothetical protein dappudraft_246411
3.7
EFX78346.1hypothetical protein dappudraft_246411
3.7
EFX78346.1hypothetical protein dappudraft_246411
3.7
EFX64858.1 hypothetical protein dappudraft_65872
6.8
EFX64858.1 hypothetical protein dappudraft_65872
6.8
EFX64858.1 hypothetical protein dappudraft_65872
6.8
EFD86972.1 hypothetical protein dappudraft_187411
15
EFX75855.1 hypothetical protein dappudraft_322949
18
P83180.1 hemocyanin B chain Pontastacus
leptodactylus
10
P83180.1 hemocyanin B chain Pontastacus
leptodactylus
10
hemocyanin (multiple)
2.00E-05
hemocyanin (multiple)
2.00E-05
EFX81277.1 hypothetical protein dappudraft_102637
3.1
ABB9165.1 calcified cuticle protein CP19.0 isoform
B Callinectes sapidus, others
2.00E-04
ACS44711.1 hemocyanin Porcellio scaber
0.058
ACS44713.1 hemocyanin subunit 2 or ACS44712.1
hemocyanin subunit 1 Eurydice pulchra
0.017
De Novo Sequenced Peptides from O. rusticus, from PEAKS Software
70
7
9.6
6.7
68
61
94
76
6.9
7.5
74
61
53
79
60
75
7.4
6.1
4.3
14.9
7.8
9.1
8
73
73
68
73
8
6.5
6.1
78
67
59
60
80
56
77
56
72
7.1
7.4
10.6
6
8
6.1
8.5
4.5
9.3
7.7
8.6
6.3
7.2
70
78
63
79
67
55
90
85
72
80
66
58
76
5.5
9
8.5
7.2
7.2
5.9
5.3
8.4
6.7
63
68
75
53
61
59
61
78
67
7
9.5
10.5
5.9
6.1
5.9
4.8
8.5
5.4
9.7
7.4
7
6.3
11.7
5.9
4.5
6
14.9
4.1
7.1
69
92
77
63
65
59
56
60
78
52
59
ALC
ALC
TLC_brain (%)_brain
ALC
TLC_brain (%)_brain
ALC
1
1
TLC_eye1 (%)_eye1 2
2
TLC_eye2 (%)_eye2
424
AABZ79135.1 arginine kinase Procambarus clarkii
EFX82438.1 hypothetical protein dappudraft_30262
EFX86228.1 hypothetical protein dappudraft_308434
EFX66745.1 hypothetical protein dappudraft_116065
ACV73257.1 cytochrome C oxidase subunit I
(Cyzicus spp WRH-2009) or ACV73163.1 same
Limnadia lenticularus
ACO15009.1 torsin-1B precursor Caligus clemensi
EFX79110.1 hypothetical protein dappudraft_245307
ABI34072.1 ATP/ADP translocase Pacifastacus
leniusculus or AEZ68611.1 adenine nucleotide
translocase Litopenaeus vannamei
ADD38275.1 probable pyruvate dehydrogenase E1
component subunit alpha, others
P05547.1 troponin I Pontastacus leptodactylus
P05547.1 troponin I Pontastacus leptodactylus
EFX59854.1 hypothetical protein dappudraft_126524,
others
BAE94239.1 aryl hydrocarbone receptor nuclear
translocator, daphnia magna, others
EFX76176.1 hypothetical protein dappudraft_107260
EFX60320.1 hypothetical protein dappudraft_278059
EFX85893.1 hypothetical protein dappudraft_98546
AEB54632.1 arg kinase Procambarus clarkii
AEB54632.1 arg kinase Procambarus clarkii
AEB54632.1 arg kinase Procambarus clarkii
AEB54632.1 arg kinase Procambarus clarkii
AEB54632.1 arg kinase Procambarus clarkii
AEB54632.1 arg kinase Procambarus clarkii
ABX44762.3 cytosolic manganese superoxide
dismutase Procambarus clarkii
EFX74602.1 hypothetical protein dappudraft_251748
EFX66222.1 hypothetical protein dappudraft_263488
EFX82754.1 hypothetical protein dappudraft_101360
EFX82754.1 hypothetical protein dappudraft_101361
ABQ41245.1 male reproductive-related LIM protein
Macrobrachium rosenbergii
EFX90150.1 hypothetical protein dappudraft_299958
EFX78266.1 hypothetical protein dappudraft_305250
EFX70769.1 hypothetical protein dappudraft_112349
ACP19741.1 thymosin-repeated protein 2 Eriocheir
sinensis and others
CAR64036.1 NADH dehydrogenase subunit 1
Catroptrus nitidus
EFX77232.1 hypothetical protein dappudraft_305825
EFX76032.1 hypothetical protein dappudraft_322770
EMQDGLLELLKLEQEM
EVTPVVAA
EVVTTKLTKK
FAGVGLATRHGT
QLLVKVVNH
QTDTPNVAAVT(-.98)
RLPDNGV
QGLEGFSPDKGG(-.98)
LSMDTGSHLEEKK
PHYGMST
Q(-17.03)LGYGGFQQPVGSYPR
QGAPDTPNADTAA
LDLNAYLAALEKKLAELSG
LEGDLPDLPKVE
LETPNKL
LGLGGGFGR
LGLGGGFGR
HLYDHLH
LAEFEHFDKF
LDAAASPAPAK
LDAAASPAPAQKATK
LDAATLAAGLEEGF
LDAATLAGALEEGFK
LDAATLAGALEEGFK
LDAATLAGALEEGFK
LDAATLAGALEEGFK
LDAATLAKLE
GSADLPGFGSV(-.98)
FNHYGMST
GDAPAAEDVKAPES
GDAPAAEDVKAPES
FLAGGLAAANLT
FAGVGLHVGAT
FAGVGLVGAT
FDDDFLTSPT
ABM54471.1 cryptocyanin Portunus pelagicus
EGFSPQTTCST
1
8.7
19
0.002
1.00E-04
10
0.059
7.3
7.00E-10
0.22
4.2
1.9
2.9
0.9
1.5
0.87
0.5
0.62
0.075
1.075
2.075
3.075
0.33
9
1.3
1.2
1.2
0.99
1.2
14
0.47
6.00E-07
1.8
10
6.5
0.5
5
8.8
55
80
55
64
10.2
6.6
81
65
73
71
70
57
9.1
11
10.7
10.5
8.6
10.5
81
73
8.9
10.2
80
88
59
74
5.9
8.9
8.8
8.8
64
10.9
4
10.2
5.3
58
79
75
70
57
53
62
6.2
13.3
6.8
3.7
72
68
59
68
77
78
74
67
61
60
76
10.8
10.2
4.1
6.8
8.5
10.9
10.4
8.1
6.1
4.8
8.3
5.2
8.1
5.7
14.1
7.9
4
4.8
4.5
9.2
11.1
4
6.6
5
10
8.8
7
4.3
7.4
58
62
81
74
66
57
53
51
66
74
58
66
63
72
73
70
54
68
7.5
6.2
10.8
11
6.3
9
10.9
6.6
6.1
8.5
9.7
8.9
5.2
8.1
11.2
69
52
67
84
63
82
73
60
76
53
88
89
52
68
66
425
YGGYFPRSPDNK
WSGPGHAPAAGGW(-.98)
VSGGSGFGHGGGFPNYGGAW(.98)
NYGGGGYGRY(-.98)
WPSGLVLSNGQNVQYRT
VVLPVTLQGA
WPNTPEDNAAYLERN(-.98)
VKAPGLLPR
VKVVNH
VSSTLSGMSLPLE(-.98)
TVSALPDPPVAA
TEEASLKDLPKVDT
TLGDTPEVAAEQAR
TLSPTVVGP
TDVPDLAA
SSGGFAAASGP(-.98)
STAGFVTRP
TDLPDLAA
TDPVVGPSGLLDPTGK
TDPVVGPSGLLDPTGK
TDPVVGPSGLLTPTGK
TDPVVGPSGLLTPTGK
TDPVVGPSGLLTPTGK
SPVGALGLLLDDGTPVQF
SEGPSGLVLSNGQNVQYRT
RPTDQNLLPSSTGAA
RLPDNGV
0.007
1.2
0.025
0.11
EFX73534.1 hypothetical protein dappudraft_325069
EFX83421.1 hypothetical protein dappudraft_315888
2.00E-04
0.84
0.55
2
31
0.62
1.9
1.00E-04
0.32
4.6
6.1
6.5
12
4.5
0.39
1.39
2.39
3.39
4.39
0.003
1.00E-06
0.099
20
EFX76032.1 hypothetical protein dappudraft_322771
AAY89318.1
glyceraldehyde-3-phosphate
dehydrogenase Tigriopus japonicus
ABM54457.1 cuticle protein CUT2 Portunus
pelagicus, other spp
ABM54460.1 cuticle protein CUT 9 Portunus
pelagicus or ABM54459.1 cuticle protein CUT8
Portunus pelagicus
ABQ59095.1 or ABQ59.96.1 chitinase Homarus
americanus
EFX62683.1 hypothetical protein dappudraft_336600
EFX87331.1 hypothetical protein dappudraft_97181
EFX70090.1 hypothetical protein dappudraft_300586
EFX70090.1 hypothetical protein dappudraft_300587
EFX70090.1 hypothetical protein dappudraft_300588
EFX70090.1 hypothetical protein dappudraft_300589
EFX70090.1 hypothetical protein dappudraft_300590
ACO14744.1 general transcription factor IIF subunit 3
Caligus clemensi
AEL23126.1 thymosin-repeated protein 1 Cherax
quadricarinatus
P81583.1 cuticle protein CP1499 Cancer pagurus
EFX86349.1 hypothetical protein dappudraft_299671
EFX62924.1 or EFX67775.1 hypothetical protein
dappudraft_336202 or 330715
EFX70812.1 hypotherical proteind appudraft_309281
or ADC55251.1 mitochondrial ATP synthase alpha
precursor Litopenaeus vannamei
3 diff hypothetical daphia prots
ACB46937.1 arg kinase Eurtium limosum and others
YP_003084316.1 cytochrome c oxidase subunit II
Metacrangonyx longipes
EFX71970.1 hypothetical protein dappudraft_308613
ABM54457.1 cuticle protein CUT2 Portunus
pelagicus
ABM74407.1 hemocyanin Portunus pelagicus and
other spp
EFX90425.1 hypothetical protein dappudraft_30013
10.2
5.4
7.9
60
54
61
61
68
56
73
69
80
79
78
11.8
11
12.9
12.7
12.4
8.6
9.5
5.1
64
65
60
70
53
7.1
5.8
10.7
13.4
7.9
10.7
5.9
4.3
6.7
4.7
5
9.1
4
71
65
72
56
58
62
61
57
7.7
10.3
5.3
3.4
7
5.4
4.4
64
69
59
56
58
68
55
10.6
5.4
8.2
6.6
9.3
5.2
7.9
9.6
9
5.6
6.7
5.2
9.5
12.1
53
54
68
51
55
52
61
68
64
63
60
58
53
63
426
Brain 1
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
Protein Description
14-3-3 [Portunus trituberculatus]
14-3-3 protein [Scylla paramamosain]
14-3-3 zeta [Artemia franciscana]
14-3-3 zeta [Fenneropenaeus merguiensis]
14-3-3 zeta [Fenneropenaeus merguiensis]
14-3-3 zeta [Scylla paramamosain]
14-3-3 zeta protein [Artemia franciscana]
14-3-3-like protein [Penaeus monodon]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
Protein Accession
gi|401816688|gb|AFQ20813.1|
gi|380003174|gb|AFD28274.1|
gi|161898814|gb|ABX80390.1|
gi|298570899|gb|ADI87601.1|
gi|298570899|gb|ADI87601.1|
gi|380042038|gb|AFD33362.1|
gi|282895616|gb|ADB03180.1|
gi|66774602|gb|AAY56092.1|
Proteins found in O. rusticus, Mascot
285
285
285
285
285
285
285
285
285
285
285
285
285
285
285
285
285
140
140
140
140
140
140
140
140
140
Protein
Mascot
Score
56
56
56
56
56
56
56
56
68.37
63.32
57.19
55.38
48.21
47.45
40.83
39.74
36.31
35.9
34.01
30.46
30.11
29.9
29.58
29.46
27.85
17.18
17.78
20.62
28.53
30.08
30.79
42.88
55.38
66.14
Peptide
Mascot
Score
55.65
55.65
55.65
55.65
15.58
55.65
55.65
55.65
QVFRRLTSAVNE
AGFAGDDAPRAVFPSI
VG
AGFAGDDAPR
TEAPLNPKANR
TEAPLNPKANR
LTEAPLNPK
AGFAGDDAPRAVF
TEAPLNPK
HQGVMVGMGQ
GDDAPRAVFPSIVG
FAGDDAPR
EAPLNPKANR
TEAPLNPK
FAGDDAPRAVFPSIVG
DDAPRAVFPSIVG
APLNPK
AGFAGDDAPRAVFPS
DEAQSKRGILT
FAPLFDPIIEDY
FAPLFDPIIED
LFAPLFDPIIED
APLFDPIIED
DVIQSGVENLDSGVG
ATLLDVIQSGVENL
ATLLDVIQSGVENLDS
GVG
EVQQKLIDDHFLFK
Peptide Sequence
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
CTLIMQLLR
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
2 Oxidation (M)
Oxidation (M)
Peptide
Variable
Modifications
0.0000400400.0
0.000040000.0
Position of Variable
Modifications
427
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
Chain A, 1.2a Resolution Structure Of A
Crayfish Trypsin Complexed With A Peptide
Inhibitor, Sgti
Chain A, 1.2a Resolution Structure Of A
Crayfish Trypsin Complexed With A Peptide
Inhibitor, Sgti
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|99032198|pdb|2F91|A
gi|99032198|pdb|2F91|A
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
152
152
152
152
152
152
152
152
152
152
42
42
167
167
285
285
285
285
285
167
167
167
167
167
167
167
167
167
285
285
285
285
20.17
18.13
24.13
27.58
30.55
32.79
33.99
34.3
67.64
79.96
23.7
44.11
17.65
18.63
17.65
16.81
16.68
15.87
15.69
57.19
55.38
48.21
45.31
40.83
34.01
30.46
29.58
24.19
18.63
26.31
24.44
21.18
EVILPVPAF
NVINGGSHAGNK
MGTEVYHHLK
NVINGGSHAGNKLA
EVILPVP
EVILPVPAFNVINGGS
HAGNKLA
EVILPVPAF
DEGGFAPNILN
AGAAELGIPLYR
DEGGFAPNILNNK
AGELDMSVNEGS
IVGGTDATLGEFPYQL
EAPLNPK
EAPLN
EAPLNPK
LKYPIE
DEAQSKRGILT
AGFAGDDAPRA
LPHAILR
TEAPLNPKANR
TEAPLNPKANR
LTEAPLNPK
DSGDGVSHTVPIYEG
TEAPLNPK
EAPLNPKANR
TEAPLNPK
APLNPK
VSHTVPIYEG
EAPLN
FAGDDAPRAVFPS
RGILTLK
LDLAGR
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
Glu->pyro-Glu
(N-term E)
Amidated (Cterm)
Glu->pyro-Glu
(N-term E)
0.4000000000.0
0.000000000000.1
3.0000000.0
0.00000.1
3.0000000.0
0.00000.1
428
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
gi|225709118|gb|ACO10405.1|
gi|225709118|gb|ACO10405.1|
gi|290784917|dbj|BAI79321.2|
gi|402715413|pdb|1PDZ|A
duplex-specific nuclease [Chionoecetes opilio]
GDP-fucose protein O-fucosyltransferase 1
precursor [Caligus rogercresseyi]
GDP-fucose protein O-fucosyltransferase 1
precursor [Caligus rogercresseyi]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
28
28
28
28
28
28
28
28
28
106
106
106
106
106
106
106
106
106
106
106
17
17
20
152
15.32
15.42
15.83
17.88
18.07
18.65
20.12
22.08
23.07
17.61
18.97
22.05
26.08
26.58
28.62
32.31
32.92
48.72
49.96
60.57
15.25
17.24
19.84
17.87
KSPNNFNLKFFN
FVPRTVMIGGK
LPAPHEP
VLYPNDNFFEGK
GANIE
VVHLDQL
VDIEGLPWAKAW
QEYFMVA
AMPYDNPIPGYK
TVDGPSAKDWR
DGPSAKDWR
TVDGPSAKDWR
TVDGPSAKDWR
GGRGAAQNIIPSS
AQNIIP
GGRGAAQNIIPSSTG
GGRGAAQNIIPS
GPSAKDWR
TVDGPSAKDWR
GPSAKDWR
HMDLII
LPNAPF
MIKAYSQSSQLV
DEGGFAPNIL
Oxidation (M)
Amidated (Cterm)
Oxidation (M)
Oxidation (M)
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
0.000000000000.1
0.00000040000.0
0.0000400.0
0.040000000000.0
0.040000.0
0.000000.1
0.000000000000.1
429
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
51.9
49.76
52.47
53.05
54.12
54.65
55.05
56.84
56.91
57.6
59.56
60.73
61.17
67.99
68.81
69.31
71.47
72.37
77.53
78.13
79.09
83.5
95.24
102.96
108.07
VFKVQNQHGQVVK
VSFPQLIHDADEAVA
DGFAPHTTYK
FAPHTTYK
FNMPPGVMEHFET
EVTPHLFTNSEVIDQA
YAAK
YGGEFPARPDNK
FNMPPGVMEHFET
DTDVAHQQQAINR
DTDVAHQQQAINRL
EVTPHLFTNSEVIDQ
NEHGIDILGDIIE
VQNQHGQVVKIFHH
DEAVANGAELPHK
YDAERLSNFLPAVDE
L
DTDVAHQQQAINR
DAERLSNFLPAVDEL
NEHGIDILGDIIESS
DTDVAHQQQAINRLL
DTDVAHQQQAINRLL
EVTPHLFTNSEVIDQA
YAAK
DTDVAHQQQAINRLL
YKVT
DTDVAHQQQAINRLL
YK
DTDVAHQQQAINRLL
YK
VSFPQLIHDADEAVA
NGAELPHKESR
2 Oxidation (M)
Amidated (Cterm)
0.0040000400000.0
0.000000000000000.1
430
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
30.83
30.88
30.98
31.41
31.54
32.5
34.02
35.01
35.24
35.59
38.54
38.59
38.68
39.24
39.8
40.24
40.42
41.36
42.35
44.23
44.6
45.01
45.39
47.05
KFNMPPGVMEHFET
VFKVQNQHGQVVK
DEAVANGAELPH
LPPLYEVTPHLF
SSESSVAIPDRVSFPQL
VQNQHGQVVKIFHH
SSESSVAIPDR
VSFPQLIHDADEAVA
NGAELPH
SSESSVAIPDRVSFPQ
YGGEFPARPDNKEF
EAAETWNPRDHTDK
GAELPHKESR
LFTNSEVIDQ
LIHDADEAVANGAEL
PH
AETWNPRDHTDK
EAAETWNPRDHTDK
VSFPQLIHDADEAVA
N
SSESSVAIPDR
EAAETWNPR
DTDVAHQQQAINRLL
ALHNQAHRVLG
NEHGIDILGDIIESS
MTQTPGNFK
VMEHFETATR
NGAELPHK
2 Oxidation (M)
Amidated (Cterm)
Oxidation (M)
Oxidation (M)
0.00040000400000.0
0.0000000000.1
0.400000000.0
0.0400000000.0
431
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
1345
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
17.9
17.85
18.55
19.28
19.82
19.94
20.13
21.22
21.39
21.76
21.82
22.29
22.95
24.32
24.35
26.46
26.53
26.6
27.23
27.46
27.88
28.55
28.79
29.83
30.62
YGGEFPARPD
NEHGIDILGDIIES
MPPGVMEHFET
DADEAVANGAELPH
ATRDPAFFRLHK
TWNPRDHTDK
NEHGIDILGDIIES
APHTTYK
ATRDPAFFRLH
KEAAETWNPRDHTD
K
EVTPHLF
KEAAETWNPRDHTD
K
ATRDPAFFR
EAAETWNPRDHTDK
DTDVAHQQQAINRLL
Y
LLLPKGK
DTDVAHQQQAINRLL
Y
LSNFLPAVDEL
GFPFDRPIPDLRV
VTPHLFTNSEVIDQ
MPPGVMEHFETATR
EVTPHLFTNSE
ATRDPAF
ETWNPRDHTDK
DPAFFR
2 Oxidation (M)
2 Oxidation (M)
Amidated (Cterm)
0.40000400000.0
0.00000000000000.1
0.40000400000000.0
432
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
1345
1345
1345
1345
1345
1345
1345
1345
1345
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
116
116
116
116
116
116
116
116
116
116
116
116
116
116
1345
hemocyanin [Pacifastacus leniusculus]
15.85
16.51
17.31
20.45
21.4
25.04
27.82
28.41
32.01
33.31
35.51
36.75
46.57
47.93
15.15
15.41
15.69
16.24
17.06
17.18
17.56
17.59
17.72
17.84
LPNRFLLP
EVIHEAYKA
KAIDE
LPNRFLLPK
TAHIMLGR
QYPDKRPHGYPLDR
EVTPHMFTNSE
TRDPSFFR
LPPGVLEHF
DPSFFR
NTAHIMLGR
EALPN
NTAHIMLGR
DPSFFRLH
RPDNKEF
EVTPHLFT
IDILGD
DGFAPHTTYKYG
EVTPHLF
KPFSLNIH
GFAPHTTYK
VSFPQLIHDA
WAHHQLTAR
PHTTYK
Amidated (Cterm)
Oxidation (M)
Glu->pyro-Glu
(N-term E)
Oxidation (M)
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
Glu->pyro-Glu
(N-term E)
Glu->pyro-Glu
(N-term E)
0.000000000.1
0.00004000.0
3.00000000000.0
0.000004000.0
0.00000.1
0.000004000.0
3.00000000.0
0.000000.1
3.0000000.0
433
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
116
116
116
17.93
17.89
17.59
18.15
18.13
18.06
18.05
17.94
18.22
18.69
18.68
18.83
18.8
20.8
20.75
19.98
19.47
19.13
19.1
21.04
20.96
23.37
22.07
23.59
23.58
26.02
24.07
23.65
36.04
28.26
26.84
15.22
15.55
15.83
RKPKTEK
LTTRLAGEVE
KEEQATSIISEHQSLTV
PDTRETIQ
KPNSLQVIDVT
QENGLAH
KVPEQV
DFIIE
AEGERCHLE
EKIEEVSEE
KPFKTDKAKEPESVS
DKPDIQKAE
HLDIP
KDDKPKE
APVDIKE
EDGTEALTLPYAVL
IPQKSVNVS
QFKLKPKTKKKVK
VTVKEIPTDI
KMKPKKKP
AVVPRGE
KPKEKVVEEI
KILDKPSQP
KAKKVMPQQESI
KPGKKPSKKI
QIKFKPGK
KKPETQQ
FEARLVPT
KPFEKPKP
PKPER
DEEKPKPEE
ALELDRF
KYGGYFPSRPDN
DPSFFRL
0.0000000.1
0.000000000.1
0.000000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
0.0000000.1
0.04000000.1
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm)
0.0000000000.1
Amidated (Cterm)
0.00000000.1
0.000000000000.1
Amidated (Cterm)
0.000000000.1
0.00000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
0.00000000.1
Amidated (Cterm)
434
16
16
16
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
inhibitor of apoptosis protein [Penaeus
monodon]
inhibitor of apoptosis protein [Penaeus
monodon]
inhibitor of apoptosis protein [Penaeus
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|133754273|gb|ABO38431.1|
gi|133754273|gb|ABO38431.1|
gi|133754273|gb|ABO38431.1|
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
25
25
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
17.58
16.13
17.87
15.1
15.16
15.12
15.12
15.41
15.29
15.42
15.42
15.44
15.65
15.5
15.48
15.69
15.67
15.65
15.7
15.89
15.89
16.6
16.36
15.92
16.68
17.25
17.23
17
16.79
17.56
17.31
IRMCKVCMD
ETRQTLQADPIESEA
KDLDPL
EFKET
EHAEVI
KRPPM
IPKTRQ
EKLDESDKK
EPEVEKRV
AKAIIADDVKDS
QTSLL
VIKPEE
EKEEVIEEV
ITQAPRFVVK
VRLIPTSDP
IDIVEG
EVVVETVA
SDPFMR
QTRTVREVQQGV
GIILEP
KAPTEEKFE
KKRILIVKS
PEEEGP
KPSEVQP
EYEARAVNK
QQQQSMSQQ
PEEIPEKE
HNETTGVVT
VQYDQNGASCLQIL
EAKPV
QSQVP
3.000000.0
0.00004.0
Glu->pyro-Glu
(N-term E)
Oxidation (M)
Glu->pyro-Glu
(N-term E)
Amidated (Cterm)
Amidated (Cterm); 2
Oxidation (M)
Glu->pyro-Glu
0.000000000.1
0.004000040.1
3.000000000000000.0
0.000000.1
3.00000.0
0.000000000000.1
2.00000.0
0.000000.1
0.000000000.1
0.000040.0
Amidated (Cterm)
Gln->pyro-Glu
(N-term Q)
Amidated (Cterm)
Amidated (Cterm)
Oxidation (M)
Amidated (Cterm)
0.000000.1
0.000000000000.1
0.000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
0.000000000.1
3.000000000.0
0.000004000.1
Amidated (Cterm);
Oxidation (M)
Glu->pyro-Glu
(N-term E)
Amidated (Cterm)
0.00000.1
Amidated (Cterm)
435
Brain 2
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
gi|321467478|gb|EFX78468.1|
gi|321467477|gb|EFX78467.1|
gi|321467477|gb|EFX78467.1|
gi|321467477|gb|EFX78467.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
Protein Description
14-3-3 protein [Scylla paramamosain]
14-3-3 protein [Scylla paramamosain]
14-3-3 zeta [Artemia franciscana]
14-3-3 zeta [Artemia franciscana]
14-3-3 zeta [Fenneropenaeus merguiensis]
14-3-3 zeta [Fenneropenaeus merguiensis]
14-3-3 zeta [Scylla paramamosain]
14-3-3 zeta [Scylla paramamosain]
14-3-3-like protein [Penaeus monodon]
14-3-3-like protein [Penaeus monodon]
ABC protein, subfamily ABCH [Daphnia pulex]
putative DH31 receptor [Daphnia pulex]
putative DH31 receptor [Daphnia pulex]
RecName: Full=NAD-dependent protein
deacylase; AltName: Full=Regulatory protein
SIR2 homolog 5; Flags: Precursor
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
Protein Accession #
gi|380003174|gb|AFD28274.1|
gi|380003174|gb|AFD28274.1|
gi|161898814|gb|ABX80390.1|
gi|161898814|gb|ABX80390.1|
gi|298570899|gb|ADI87601.1|
gi|298570899|gb|ADI87601.1|
gi|380042038|gb|AFD33362.1|
gi|380042038|gb|AFD33362.1|
gi|66774602|gb|AAY56092.1|
gi|66774602|gb|AAY56092.1|
gi|321467478|gb|EFX78468.1|
gi|225908473|gb|ACO36738.1|
gi|225908473|gb|ACO36738.1|
gi|225908473|gb|ACO36738.1|
gi|225908473|gb|ACO36738.1|
gi|225908473|gb|ACO36738.1|
gi|225908473|gb|ACO36738.1|
gi|387935383|sp|E9GD30.1|SIR
5_DAPPU
gi|321458499|gb|EFX69566.1|
gi|321458499|gb|EFX69566.1|
gi|83638451|gb|ABC33915.1|
gi|133754273|gb|ABO38431.1|
monodon]
inhibitor of apoptosis protein [Penaeus
monodon]
kazal-type proteinase inhibitor [Fenneropenaeus
chinensis]
97
97
97
31
17
31
31
39
Protein
Mascot
Score
106
106
106
106
106
106
106
106
106
106
17
39
39
39
39
39
43
32
32
24
16
27.67
52.96
70.36
15.04
17.46
31.05
22.81
15.34
Peptide
Mascot
Score
66.45
64.1
66.45
64.1
66.45
64.1
66.45
64.1
66.45
64.1
22.81
15.89
17.92
22.6
23.86
30.44
43.47
31.97
16.08
23.85
15.24
ASVHIKLP
ATLLDVIQSGVENL
NTTIVQVPF
ATLLDVIQSGVENLDS
GVG
DLPKLAAPSSQ
FLLPAA
IPGPRVGYMP
Peptide Sequence
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
ASNPESKVFYLK
IPGPRVGYMP
VMNKFMAGLGVPES
VRWLPLESNPAVMNK
EEQEAAGGDVPE
VRWLPLE
VRWLPLESNPAVMNK
VRWLPLESNPAVMNK
F
IAEFEHRMT
QSLKCTR
LPAVVT
KHDGPCAAKVI
PSTSGAQA
Oxidation (M)
Amidated (Cterm)
Oxidation (M)
Amidated (Cterm)
Oxidation (M)
Peptide
Variable
Modifications
Oxidation (M)
Oxidation (M)
Oxidation (M)
Oxidation (M)
Amidated (Cterm)
(N-term E)
0.000000000.1
0.0000000040.0
0.00000000000.1
0.0000000040.0
Position of Variable
Modifications
0.00000400000000.0
0.000000000000400.0
0.0000000000004000.0
0.000000000000400.0
0.000000040.0
0.0000000.1
436
caspase 1, partial [Daphnia parvula]
caspase 5, partial [Daphnia pulex]
caspase 5, partial [Daphnia pulex]
gi|321478601|gb|EFX89558.1|
gi|385048432|gb|AFI39977.1|
gi|385048432|gb|AFI39977.1|
gi|385048432|gb|AFI39977.1|
gi|385048432|gb|AFI39977.1|
gi|385048472|gb|AFI39997.1|
gi|385048472|gb|AFI39997.1|
21
21
15
23
15
15
15
Btk family kinase at 29A-like protein [Daphnia
pulex]
caspase 1, partial [Daphnia parvula]
caspase 1, partial [Daphnia parvula]
caspase 1, partial [Daphnia parvula]
gi|321478601|gb|EFX89558.1|
281
281
127
127
127
127
127
127
127
127
97
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
281
23
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
Btk family kinase at 29A-like protein [Daphnia
pulex]
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|211947526|gb|ACJ13518.1|
gi|551380|gb|AAB31477.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
21.58
20.87
15.39
18.89
22.5
19.53
15.74
23.18
17.14
16.48
72.03
48.24
44.53
37.2
34.87
24.06
19.89
17.78
27.4
72.03
66.87
57.89
48.24
47.87
43.42
42.27
41.74
37.2
35.54
34.87
34.43
32.21
27.21
26.61
25.84
24.06
22.77
22.62
19.89
17.78
17.33
KMMKEGTM
LDAGTTMLPP
LDAGTTMLPP
LPNVTET
QGDRLDAGTTMLPKV
TETDS
DLLNKLINVLNDLQF
N
DLLNKLINVLNDLQF
SIPTEPAVVP
RGILTLK
FAGDDAPR
TEAPLNPKANR
EAPLNPKANR
VSHTVPIYEG
APLNPKANR
TEAPLNPK
EAPLNPK
TEAPLNPK
TEAPLNPK
LFAPLFDPIIED
TEAPLNPKANR
AGFAGDDAPR
HQGVMVGMGQK
EAPLNPKANR
AGFAGDDAPR
FAGDDAPR
AGFAGDDAPR
AGFAGDDAPR
APLNPKANR
DAPRAVFPSIVG
TEAPLNPK
AGFAGDDAPRAVFPS
GFALPHAILR
AGFAGDDAPRA
DEAQSKRGILT
FAGDDAPRAVFP
EAPLNPK
LVVDNGSGMVK
VVDNGSGMVK
TEAPLNPK
TEAPLNPK
LKYPIE
Amidated (Cterm)
Amidated (Cterm)
Amidated (C-
Amidated (Cterm);
Oxidation (M)
Oxidation (M)
Oxidation (M)
Amidated (Cterm)
0.0000000000000000.1
0.00000000000000000.
0.000000000000000000
00.1
0.00000004.1
0.0000004000.0
0.0000004000.0
0.0000000.1
437
caspase 5, partial [Daphnia pulex]
caspase 5, partial [Daphnia pulex]
caspase 5, partial [Daphnia pulex]
caspase 5, partial [Daphnia pulex]
caspase 5, partial [Daphnia pulex]
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
gi|385048478|gb|AFI40000.1|
gi|385048478|gb|AFI40000.1|
gi|385048464|gb|AFI39993.1|
gi|385048464|gb|AFI39993.1|
gi|385048464|gb|AFI39993.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743896|dbj|BAJ23879.1|
gi|310743900|dbj|BAJ23881.1|
gi|310743900|dbj|BAJ23881.1|
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
fructose 1,6-bisphosphatase [Marsupenaeus
japonicus]
fructose 1,6-bisphosphatase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
glycogen phosphorylase [Marsupenaeus
japonicus]
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
caspase 5, partial [Daphnia pulex]
gi|385048478|gb|AFI40000.1|
gi|402715413|pdb|1PDZ|A
caspase 5, partial [Daphnia pulex]
gi|385048472|gb|AFI39997.1|
29
29
29
29
29
29
28
28
97
97
97
97
97
97
97
97
21
21
21
21
21
21
21
15.02
16.03
18.5
22.31
23.39
29.54
16.77
28.02
16.68
18.54
19.1
19.18
27.67
28.58
41.71
82.06
20.48
20.87
21.58
20.48
20.87
21.58
20.48
SLAIPEL
LPAPHEP
ILPRHLQ
TNGITPR
RDIFKDF
NNVVNTMRL
KNNEAVPSVSDALQ
MAGDVNVQ
SITKVFART
LAKYNQILRIEEELGS
GA
KDFPI
PIVSIEDPFDQ
MILPTGA
DEGGFAPNILN
DEGGFAPNILNNK
DEGGFAPNILNNK
KV
DLLNKLINVLNDLQF
KV
DLLNKLINVLNDLQF
N
DLLNKLINVLNDLQF
KV
DLLNKLINVLNDLQF
KV
DLLNKLINVLNDLQF
N
DLLNKLINVLNDLQF
KV
DLLNKLINVLNDLQF
KV
Amidated (Cterm)
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
term)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
0.0000000.1
0.40000000.1
0.4000000.1
0.0000000000000000.1
0.00000000000000000.
1
0.00000000000000000.
1
0.0000000000000000.1
0.00000000000000000.
1
0.00000000000000000.
1
1
0.00000000000000000.
1
438
25
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
glycogen synthase [Marsupenaeus japonicus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
gi|310743898|dbj|BAJ23880.1|
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
25
25
glycogen synthase [Marsupenaeus japonicus]
glycogen synthase [Marsupenaeus japonicus]
gi|310743898|dbj|BAJ23880.1|
gi|310743898|dbj|BAJ23880.1|
42.2
41.86
42.99
44.14
44.31
44.47
45.69
47.65
54.3
56.62
58.85
60.11
61
62.14
62.87
68.02
70.13
72.32
73.5
74.67
77.72
83.48
15.9
25.11
18.49
VMEHFETATR
EAAETWNPRDHTDK
ATRDPAFFRLH
DTDVAHQQQAINR
VFKVQNQHGQVVK
SSESSVAIPDR
RPMGFPFDRPIPDLR
AETWNPRDHTDK
DTDVAHQQQAINRLL
DTDVAHQQQAINRLL
DTDVAHQQQAINR
VFKVQNQHGQVVKIF
HH
DTDVAHQQQAINRLL
Y
SSESSVAIPDRVSFPQ
VQNQHGQVVKIFHH
EAAETWNPRDHTDK
FNMPPGVMEHFETAT
RDPAF
DTDVAHQQQAINR
LIHDADEAVANGAEL
PHK
KEAAETWNPRDHTD
K
KEAAETWNPRDHTD
K
DTDVAHQQQAINRLL
Y
EKINDFIRG
EASVRMEVE
LTTAINNIR
0.000004000.1
0.000000000.1
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm)
439
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
24.52
24.61
24.73
24.99
25.03
25.13
25.86
29.74
29.74
30.42
30.72
31.14
31.94
32.88
33.17
33.89
33.95
35.32
38.56
39.81
39.94
40.06
40.86
41.66
LLLPKGK
EVTPHLFTNS
LIHDADEAVANGAEL
PHK
MEFTGSKKNR
GEFPARPDNK
ATRDPAFFRLHK
DAERLSNFLPAVDEL
NEHGIDILGDIIES
PFDRPIPDLR
FPQLIHDADEAVAN
EAAETWNPR
AERLSNFLPAVDEL
EVTPHLFTNSEVID
DPAFFRLH
LSNFLPAVDEL
AETWNPRDHTDK
ETWNPRDHTDK
KFNMPPGVMEHFETA
TRDPAF
KEAAETWNPRDHTD
K
EVTPHLFTNSE
EAAETWNPRDHTDK
MPPGVMEHFET
TDLKEAAETWNPRDH
TDK
EAAETWNPRDHTDK
Glu->pyro-Glu
(N-term E)
Glu->pyro-Glu
(N-term E)
3.0000000000.0
3.00000000000000.0
440
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
994
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
110
110
110
110
994
hemocyanin [Pacifastacus leniusculus]
34.79
34.2
37.6
42.73
15.08
15.44
15.73
15.86
16.04
16.63
16.98
18.3
18.32
19.07
19.08
19.26
19.49
20.21
20.71
21.31
21.53
21.53
21.82
23.01
23.92
YMDNIFR
LPPGVLEHFET
EVTPHMFTNSE
LHNTAHIMLGR
GFPFDRP
VTPHLFT
PFDRPIPDLRV
EVTPHLF
AAETWNPRDHTDK
QVVKIFHH
VSFPQLIHDADEAVA
NGAELPH
VFKVQNQHGQVVK
EVTPHLFTNSEVID
VFKVQNQHGQVVKIF
HH
VSFPQLIHDADEAVA
NGAELPHK
LFTNSEVIDQ
RPMGFPFDRPIPDLRV
DVAHQQQAINR
NQHGQVVKIFHH
QNQHGQVVKIFHH
LLLPKGK
YDAERLSNFLPAVDE
L
AERLSNFLPAVDEL
FNMPPGVMEHFET
VSFPQLIHDADEAVA
N
Glu->pyro-Glu
(N-term E)
Glu->pyro-Glu
(N-term E)
Amidated (Cterm)
3.00000000000.0
3.0000000.0
0.00000000000000.1
441
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
110
110
110
110
110
110
110
110
17.76
17.47
18.2
18.8
18.95
18.81
19.09
19.07
19.18
21.61
21.35
20.63
21.89
21.73
23.23
23.37
24.5
27.11
33.95
16.58
17.89
23.75
25.63
26.61
28.07
29.51
32.17
EYEARAVNK
PLQDPNPLPE
AVSIHE
EKLDESDKK
DKPDIQKAE
KDEVE
KPFEKPKP
HNETTGVVT
KEIPEVKE
PEVPKPVE
LMVSSKES
HNETTGVVT
KEEVKEEIILK
IKAPLWRVKKLP
EKLDESDKK
QIKFKPGK
APSAPFRPH
LLLPQDSIAV
KPFEKPKP
KYGGYFPSRPDN
QYPDKRPHGYPLDR
YMDNIFREHK
GYPLDR
QYPDKRPHGYPLDR
NTAHIMLGR
LPNRFLLPK
LPPGVLEHF
0.00000000.1
0.04000000.0
Amidated (Cterm)
Oxidation (M)
3.000000000.0
0.0000000000.1
0.000000.1
0.000000000.1
0.000000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Glu->pyro-Glu
(N-term E)
Amidated (C-
0.00000000.1
0.00000000.1
Amidated (Cterm)
Amidated (Cterm)
0.000000000.1
0.00000000.1
0.000000000.1
0.0000000000.1
0.00000000000.1
0.00000000.1
0.000004000.0
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Oxidation (M)
442
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
JHE-like carboxylesterase 1 [Pandalopsis
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|326579691|gb|ADZ96217.1|
33
21
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
15.25
21.26
15.31
15.48
15.36
15.61
15.6
15.61
15.67
15.73
15.91
15.88
15.99
16.01
16.38
16.19
16.12
16.09
16.45
16.54
16.75
16.58
16.56
16.96
17
17.09
17.16
17.36
17.3
KHEDIEE
MKLLFLCVAIATWLG
KIEEVTEEVTIKKP
KPEDEE
TKIAQLE
QEVTVKGVPAD
EKIKELEDT
ADVAPSIAPLE
GIILEP
KPGKKP
TLLIIEAVPEDSA
RVEPRN
QNKLQEKI
QRVSVIVS
MQQMVSPKF
QCRAVGTPTP
TVQEVAVKE
AGQYTVVAR
KPTKIIEPE
EGLEKIRQLE
QGRAGLEKVE
QENGLAH
KRPPM
QLEEPVKP
QATSVISPLE
QPDILPGI
KDEVEPH
NKTGEA
KPSEVQP
0.000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (C-
0.00000000000.1
0.0000000.1
0.0000000000000000.1
0.00000000000000.1
0.00000000000.1
0.000000.1
0.000000.1
0.0000000000000.1
0.00000000.1
0.00000000.1
0.000400000.1
0.000000000.1
0.0000000000.1
0.00004.0
0.0000000000.1
0.00000000.1
2.0000000000.0
0.00000000.1
0.0000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
Gln->pyro-Glu
(N-term Q)
Amidated (Cterm)
Amidated (Cterm)
term)
443
Eyestal
k1
phantom [Marsupenaeus japonicus]
putative G-protein coupled receptor, orphan
[Daphnia pulex]
putative G-protein coupled receptor, orphan
[Daphnia pulex]
RecName: Full=NAD-dependent protein
deacylase; AltName: Full=Regulatory protein
SIR2 homolog 5; Flags: Precursor
RecName: Full=NAD-dependent protein
deacylase; AltName: Full=Regulatory protein
SIR2 homolog 5; Flags: Precursor
ubiquitin carboxyl-terminal esterase L3 [Scylla
paramamosain]
vitellogenin fused with superoxide dismutase
[Daphnia magna]
vitellogenin fused with superoxide dismutase
[Daphnia magna]
vitellogenin fused with superoxide dismutase
[Daphnia magna]
vitellogenin fused with superoxide dismutase
[Daphnia magna]
vitellogenin fused with superoxide dismutase
[Daphnia pulex]
vitellogenin fused with superoxide dismutase
[Daphnia pulex]
vitellogenin fused with superoxide dismutase
[Daphnia pulex]
vitellogenin fused with superoxide dismutase
[Daphnia pulex]
vitellogenin fused with superoxide dismutase
[Daphnia pulex]
vitellogenin fused with superoxide dismutase
[Daphnia pulex]
gi|224471277|dbj|BAH24005.1|
gi|224471277|dbj|BAH24005.1|
gi|46398235|gb|AAS91795.1|
gi|225712206|gb|ACO11949.1|
Protein Accession #
gi|321465377|gb|EFX76379.1|
gi|321465377|gb|EFX76379.1|
gi|321465377|gb|EFX76379.1|
gi|321465377|gb|EFX76379.1|
gi|321465377|gb|EFX76379.1|
gi|321465377|gb|EFX76379.1|
gi|95113632|dbj|BAE94322.1|
gi|95113632|dbj|BAE94322.1|
gi|95113632|dbj|BAE94322.1|
gi|95113632|dbj|BAE94322.1|
gi|225908473|gb|ACO36738.1|
gi|387935383|sp|E9GD30.1|SIR
5_DAPPU
gi|387935383|sp|E9GD30.1|SIR
5_DAPPU
gi|321477804|gb|EFX88762.1|
gi|321477804|gb|EFX88762.1|
Protein Description
Serine/threonine-protein phosphatase 2A
regulatory subunit B [Lepeophtheirus salmonis]
intestinal trypsin 4 precursor [Lepeophtheirus
salmonis]
phantom [Marsupenaeus japonicus]
gi|225713512|gb|ACO12602.1|
gi|326579691|gb|ADZ96217.1|
japonica]
JHE-like carboxylesterase 1 [Pandalopsis
japonica]
Peptidyl-tRNA hydrolase 2, mitochondrial
precursor [Lepeophtheirus salmonis]
24
26
28
Protein
Mascot
Score
28
28
28
28
28
25
25
25
25
55
43
43
37
37
19
19
20
21
23.58
25.59
15.54
Peptide
Mascot
Score
16.15
17.32
20.25
21.2
27.98
17.03
20.25
21.2
25.21
54.57
22.28
43.58
28.78
40.82
17.79
18.88
20.5
17.03
EFTPTGE
VDEIPLEEQSQ
Peptide Sequence
EVKNTANQIT
VGEQAVQAISALVP
VRIIGCTSAEG
GARAQETVEKKLG
QRIKSI
MEGSVTGECET
KIVTVLL
GARAQETVEKKLG
QRIKSI
ASPSTIRQ
VRWLPLESNPAVM
IAEFEHRMT
IAEFEHRMT
HSIQITSTGGD
AALLDRL
LLLALLR
VVGIGPGPLE
APHLTLTNLVEKYGR
VYSLKMGGVSVVVIA
DPDLI
KDVILVV
V
Amidated (Cterm); Glu-
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Glu->pyro-Glu
(N-term E)
Peptide
Variable
Modifications
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Oxidation (M)
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
term)
Amidated (Cterm)
3.0000000.1
Position of Variable
Modifications
3.0000000000.0
0.00000000000000.1
0.00000000000.1
0.0000000000000.1
0.000000.1
0.40000000000.0
0.0000000.1
0.0000000000000.1
0.000000.1
0.00000000.1
0.000000040.0
0.000000040.0
0.00000000000.1
0.0000000.1
0.0000000.1
444
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
110
15.88
15.83
16.4
16.35
16.16
16.02
15.88
17.15
16.94
16.91
17.38
17.29
17.22
17.83
17.73
18.82
18.71
18.05
19.53
18.94
19.55
21.35
20
19.97
23.54
22.55
22.22
21.65
21.42
30.07
64.48
78.22
VVLQSQVPK
EGMPSI
IEDKAKTVLSQRVS
KSVKRVNQK
VTSASVKVT
LEPLKPLK
DKYLMTQ
QHIRSVEEM
SQIPK
KTEQAKSILSPRE
LPAPIEE
QENGLAH
SDPFMR
ERTKPEKEE
ISQDTVQVQEI
KEAPYSMEED
KDSITVTEKIP
ISQDTVQVQEI
LEAQVQPA
QLEMDVRLVAIP
KSPLE
KPFEKPKP
LEPLKPLK
AAPIEEKF
KAKKVMPQQESI
KPRFLTKLK
LSQNMH
QMQIQVAA
TVQEVSVKEAP
KRILIVKSV
IIEPEPEPEPELIILRPV
E
IIEPEPEPEPELIILRPV
ER
0.0000004000.1
0.000000000.1
0.0000000.1
Amidated (Cterm)
Amidated (Cterm)
0.000000000.1
3.000000.0
0.0000400.0
0.00000000000000.1
Amidated (Cterm)
Oxidation (M)
Amidated (Cterm)
Glu->pyro-Glu
2.000000004.0
0.000040.0
Oxidation (M)
Gln->pyro-Glu
(N-term Q);
Oxidation (M)
0.00000000.1
0.000400000000.0
0.00000.1
0.00000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
0.000000000000.1
0.000000000.1
Amidated (Cterm)
Amidated (Cterm)
>pyro-Glu (Nterm E)
445
110
110
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
510
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
110
110
110
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
18.68
19.66
23.45
25.81
26.17
28.21
30.52
37.63
38.39
42.19
42.62
45.16
46.67
48.06
61.82
62.06
67.74
68.3
83.43
99.5
107.57
15.12
15.04
15.8
15.63
15.31
RPMGFPFDRPIPDLR
DLKELESR
EVTPHL
LLLPKGK
PMGFPFDRPIPDL
VFKVQNQHGQVV
DEAVANGAELPHK
VFKVQNQHGQVVK
RPMGFPFDRPIPDLR
YGGEFPARPDN
DGFAPHTTYK
VFKVQNQHGQVVK
DTDVAHQQQAIN
EAAETWNPR
DTDVAHQQQAINR
YGGEFPARPDNK
DTDVAHQQQAINR
DTDVAHQQQAINRLL
YK
SVAIPDRVSFPQLIHD
ADEAVAN
SSESSVAIPDRVSFPQL
IH
DTDVAHQQQAINR
QEVSVKEAPTE
AAPIEEKFEIPKITLK
AGEKI
GESQYEDTVLKMQY
DAVVI
Oxidation (M)
Amidated (Cterm)
(N-term E)
0.004000000000000.0
0.00000000000.1
446
510
510
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
ecdysteroid receptor [Carcinus maenas]
ecdysteroid receptor [Carcinus maenas]
Dpahnia pulex DNMT3 [Daphnia pulex]
Dpahnia pulex DNMT3 [Daphnia pulex]
Dpahnia pulex DNMT3 [Daphnia pulex]
Dpahnia pulex DNMT3 [Daphnia pulex]
crustin-like peptide type 5 [Marsupenaeus
japonicus]
crustin-like peptide type 5 [Marsupenaeus
japonicus]
crustin-like peptide type 4 [Marsupenaeus
japonicus]
crustin-like peptide type 4 [Marsupenaeus
japonicus]
crustin-like peptide type 2 [Marsupenaeus
japonicus]
crustin-like peptide type 2 [Marsupenaeus
japonicus]
crustin-like peptide [Marsupenaeus japonicus]
crustin-like peptide [Marsupenaeus japonicus]
chymotrypsin-like protein [Daphnia pulex]
chymotrypsin-like protein [Daphnia pulex]
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, The 2.1a Crystal Structure Of Copgfp
Chain A, The 2.1a Crystal Structure Of Copgfp
caspase 6, partial [Daphnia pulex]
gi|62701385|dbj|BAD95643.1|
gi|40748295|gb|AAR89628.1|
gi|40748295|gb|AAR89628.1|
gi|321467881|gb|EFX78869.1|
gi|321467881|gb|EFX78869.1|
gi|321467881|gb|EFX78869.1|
gi|321467881|gb|EFX78869.1|
gi|402715413|pdb|1PDZ|A
gi|114793939|pdb|2G3O|A
gi|114793939|pdb|2G3O|A
gi|385048340|gb|AFI39931.1|
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
gi|46240812|dbj|BAD15063.1|
gi|46240810|dbj|BAD15062.1|
gi|46240810|dbj|BAD15062.1|
gi|321468614|gb|EFX79598.1|
gi|321468614|gb|EFX79598.1|
gi|46240812|dbj|BAD15063.1|
gi|46240816|dbj|BAD15065.1|
gi|46240816|dbj|BAD15065.1|
gi|46240818|dbj|BAD15066.1|
gi|46240818|dbj|BAD15066.1|
gi|62701385|dbj|BAD95643.1|
510
hemocyanin [Pacifastacus leniusculus]
62
20
20
25
62
62
62
34
34
34
21
21
34
34
34
34
34
38
38
27
27
38
38
25
25
510
hemocyanin [Pacifastacus leniusculus]
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
19.58
21.94
17.24
24.72
19.72
39.53
47.01
19.03
33.72
19.03
21.28
18.9
33.72
19.03
33.72
19.03
33.72
17.43
19.12
26.99
16.1
38.1
23.26
24.93
26
15.42
16.22
17.6
17.63
IEEELGSGA
MFFYHFGT
SHMHFKSAIHPSI
QSKATQTDGP
DDLTVTNPK
FAGKNFR
GNPTVEVDLYTSK
GFGGVQGGGV
GGGFGGGFGGPQGGG
GFGGVQGGGV
QSISPVLMKVT
SELFPRA
GGGFGGGFGGPQGGG
GFGGVQGGGV
GGGFGGGFGGPQGGG
GFGGVQGGGV
GGGFGGGFGGPQGGG
EADGLL
FTDVGNMSRTD
TVDGPSAKDWR
DYPSHGSPIAEEKAIPT
SPM
HITEMTILTVQLI
SLFDGIGT
SLFDGIGT
GGRGAAQNIIPSS
NMPPGV
FNMPPGVMEHFETAT
RDPAFF
NEHGIDILGDIIE
AETWNPR
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
Glu->pyro-Glu
(N-term E)
Oxidation (M)
Oxidation (M)
Oxidation (M)
Oxidation (M)
0.00000004000.0
3.000000.0
0.00000040000.1
0.000000000000000000
04.0
0.0000400000000.0
0.040000.0
0.004000000000000000
000.0
447
caspase 3, partial [Daphnia pulex]
caspase 3, partial [Daphnia pulex]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
gi|385048368|gb|AFI39945.1|
gi|385048368|gb|AFI39945.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
caspase 6, partial [Daphnia pulex]
caspase 3, partial [Daphnia pulex]
gi|385048340|gb|AFI39931.1|
gi|385048368|gb|AFI39945.1|
477
477
477
477
477
477
477
477
477
477
477
477
477
477
477
477
477
477
477
59
59
23
59
59
59
23
25
23
19.87
19.51
27.92
28.57
28.83
29.7
33.62
34.68
35.69
40.05
42.86
43.92
44.03
65
71.62
76.95
94.4
95.08
116.08
25.33
25.07
16.23
38.3
35.75
25.56
19.19
16.29
23.04
MGLTEFQAVK
QTDKHPNKDF
GTYFPLTGMSK
LIDDHFLFK
RMGLTEFQAVK
ATLLDVIQSGVENLDS
GVGIYAPDA
ASVHIKLPK
RMGLTEFQAV
ATLLDVIQSGVENLDS
GVGIYAPDAEAY
RMGLTEFQAVK
LFDPIIEDYH
FDPIIEDYH
ATLLDVIQSGVENL
ATLLDVIQSGVENLD
LIDDHFLFKEGDR
APLFDPIIEDYH
PFSSXXCATLAGKP
AVFPSIVGR
DAYVGDEAQSK
FAGDDAPRA
AGFAGDDAPRAVFPSI
VG
HQGVMVGMGQ
ATLLDVIQSGVENLDS
GVGIYAPDA
ATLLDVIQSGVENLDS
GVG
EVQQKLIDDHFLFKE
GDR
TLAGKP
EIKTIVNDL
SNSST
Oxidation (M)
Gln->pyro-Glu
Oxidation (M)
2 Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
0.4000000000.0
2.0000000000.0
0.04000000000.0
0.0000400400.0
0.00000000000000.1
0.000000.1
448
Eyestal
k2
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
ABC protein, subfamily ABCH [Daphnia pulex]
gi|551380|gb|AAB31477.1|
gi|321473807|gb|EFX84774.1|
gi|321473807|gb|EFX84774.1|
gi|321473807|gb|EFX84774.1|
gi|321473807|gb|EFX84774.1|
gi|321473807|gb|EFX84774.1|
gi|321473807|gb|EFX84774.1|
gi|321473807|gb|EFX84774.1|
Protein Description
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
Protein Accession #
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
ABC protein, subfamily ABCH [Daphnia pulex]
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
28
28
Protein
Mascot
Score
28
28
28
28
477
28
477
477
20.26
19.82
18.69
20.42
20.28
22.34
21.71
21.44
21.44
21.42
22.58
25.41
26.39
26.27
26.27
25.86
28.33
26.79
32.88
16.46
16.1
Peptide
Mascot
Score
17.07
17.78
21.13
19.33
15.3
28.33
15.51
18.05
IELTYE
KNLSNLEEGEIA
AAPIEEKF
KITPPSKPNSL
KEEITEEVIIKKPE
EKIEEVSEE
KILDKPSQP
LEVSETIVL
MSGEPTKPKF
KIVGKTATLTVNAQ
LERAKPN
KPITIGSRFKTY
LEAQVQPA
IVIKPEE
IVIKPEE
IVIKPEE
KPFEKPKP
PEREKVPDQVPKPEK
E
AAPIEEKFEIPK
Peptide Sequence
QANAIGLMRSGRL
GASGCGKTTLLSCLV
G
NGIDMSVKKGT
QANAIGLMRSGRL
QQMELNLQH
VEFITPGIILT
FAPLFDPIIE
ATLLDVIQSGVENLDS
GVGIYAPDAEAY
VEFITPGIILT
DDHFLFK
0.00000000.1
Amidated (Cterm)
0.000000000.1
0.4000000000.0
0.00000000000.1
0.000000.1
Oxidation (M)
Amidated (Cterm)
Amidated (Cterm)
0.0000000.1
0.000000000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
0.00000000.1
Position of Variable
Modifications
0.0000000000000000.1
0.00004000000.0
0.0000000000000.1
0.0000000000000.1
2.004000000.0
Oxidation (M)
Peptide
Variable
Modifications
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Gln->pyro-Glu
(N-term Q);
Oxidation (M)
(N-term Q)
449
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|249511|gb|AAB22190.1|
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
gi|249511|gb|AAB22190.1|
32
32
32
32
32
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
586
586
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
37
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
37
32
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
37
37
37
37
37
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
hemocyanin subunit c [Panulirus
interruptus=spiny lobster, Peptide, 661 aa]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|249511|gb|AAB22190.1|
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
gi|15425681|dbj|BAB64297.1|
103.33
83.53
17.36
19.76
20.09
29.39
30.96
35.29
15.05
15.61
15.39
16.04
16.02
16.01
15.78
15.62
16.18
17.4
17.2
16.83
16.69
16.63
16.23
17.67
17.76
17.83
17.77
18.19
18.09
17.89
DTDVAHQQQAINR
SSESSVAIPDRVSFPQL
QYPDKRPHGYPLDR
LPKGQAQG
VVLPPL
LMMHRVLMN
YGGYFPSRPDN
DPSFFR
QGVQRVQGPTQ
QQAPHEAVEP
LGLSDALEVS
KPEEKIE
PEEKVVEV
ILDKPSQP
DAEPPAFNPPLTP
LEEHYP
EEKVKVTPKP
QMINQGIETEE
KIAEEVTEEIVVKKA
KTEQAKSILSPRE
ENEEFLDEFIET
REWSPVP
KLKKSTRMLEPKEP
KITPPSKPNSL
LEVSSELAPD
KAKKVMPQQESI
SVHEISVKDAP
KSIISPHESLTVE
EEVKPKTEQ
VTIKQE
2.00000000000.1
Amidated (Cterm); Gln>pyro-Glu (Nterm Q)
0.040000040.1
2.0000000000.0
Gln->pyro-Glu
(N-term Q)
Amidated (Cterm); 2
Oxidation (M)
0.0000000.1
0.0000000000.1
0.04000000000.1
0.00000000000.1
0.0000000000.1
0.000000000000.1
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm)
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm)
450
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
AF522504_1
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
586
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
586
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
28.96
29.92
29.97
30.37
30.88
32.87
32.93
33.04
33.55
34.05
35.21
37.06
37.5
39.14
41.65
43.36
44.18
44.97
52.96
53.58
62.05
64.41
83.49
MGFPFDRPIPDL
VFKVQNQHGQVVKIF
HH
VSFPQLIHDA
DPAFFR
RPMGFPFDRPIPDLR
YGGEFPARPDNK
VFKVQNQHGQVVK
DTDVAHQQQAINRLL
YK
RPMGFPFDRPIPDL
VSFPQLIHDADEAVA
NGAELPH
DGFAPHTTYK
RPMGFPFDRPIPDL
DTDVAHQQQAIN
FNMPPGVMEHFETAT
RDPAFF
YPDKRPMGFPFDRPIP
DLR
FNMPPGVMEHFE
KFNMPPGVMEHFETA
TRDPAFFR
EAAETWNPR
YGGEFPARPDNK
DTDVAHQQQAINRLL
YK
FNMPPGVMEHFETAT
RDPAFF
IHDADEAVANGAELP
HK
FNMPPGVMEHFETAT
RDPAFFR
SSESSVAIPDRVSFPQL
IHDADEAVANGAELP
HKESR
Oxidation (M)
0.004000000000000000
000.0
451
586
586
586
586
586
586
586
586
586
586
586
586
586
586
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase
[Marsupenaeus japonicus]
Dpahnia pulex DNMT3 [Daphnia pulex]
Dpahnia pulex DNMT3 [Daphnia pulex]
Dpahnia pulex DNMT3 [Daphnia pulex]
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
gi|62701385|dbj|BAD95643.1|
gi|321467881|gb|EFX78869.1|
gi|321467881|gb|EFX78869.1|
gi|321467881|gb|EFX78869.1|
gi|402715413|pdb|1PDZ|A
gi|62701385|dbj|BAD95643.1|
gi|62701385|dbj|BAD95643.1|
21
42
42
22
42
22
22
22
586
hemocyanin [Pacifastacus leniusculus]
gi|62701385|dbj|BAD95643.1|
586
hemocyanin [Pacifastacus leniusculus]
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
gi|21914372|gb|AAM81357.1|
AF522504_1
21
19.21
21.93
15.28
41.93
19.65
22.31
29.19
16.68
16.9
19.76
19.96
20.09
20.63
21.49
21.55
22.33
22.79
22.85
23.17
23.3
23.61
28.09
28.26
AGAAELGIPLYR
FTDVGNMSRTD
FTDVGNMSRTD
HKGEVKAEDGC
SLFDGIGT
DGPSAKDWR
TVDGPSAKDWR
QGGAKKVI
LPKGKEQ
DTDVAHQQQAINRLL
Y
FNMPPGVMEHFETAT
RDPAFFR
VVLPPL
FNMPPGVMEHFETAT
RDPAFF
DLKELESR
TATRDPAFF
YGGEFPARPDNKEF
VQNQHGQVVKIFHH
FNMPPGVMEHFETAT
R
SSESSVAIPDRVSFPQL
IHDA
VSFPQLIHDADEAVA
NGAELPHKESR
YPDKRPMGFPFDRPIP
DL
PMGFPFDRPIPDL
YGGEFPARPD
SSESSVAIPDRVSFPQL
IH
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm)
Oxidation (M)
Amidated (Cterm)
2 Oxidation (M)
Amidated (Cterm)
0.00000040000.1
0.00000040000.1
0.00000000000.1
0.00000000.1
0.000000040000000000
0000.0
0.0000000.1
0.004000040000000000
000.0
452
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
Chain A, X-Ray Structure And Catalytic
Mechanism Of Lobster Enolase
caspase 5, partial [Daphnia pulex]
caspase 4, partial [Daphnia parvula]
caspase 4, partial [Daphnia parvula]
caspase 4, partial [Daphnia parvula]
caspase 4, partial [Daphnia parvula]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
gi|402715413|pdb|1PDZ|A
gi|385048476|gb|AFI39999.1|
gi|385048398|gb|AFI39960.1|
gi|385048412|gb|AFI39967.1|
gi|385048398|gb|AFI39960.1|
gi|385048412|gb|AFI39967.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|254832574|gb|ACT82962.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|402715413|pdb|1PDZ|A
gi|402715413|pdb|1PDZ|A
454
454
454
454
454
454
454
454
454
454
454
77
15
77
77
77
77
77
77
77
15
15
15
21
27
21
21
42.85
40.67
45.1
46.82
49.71
51.69
56.59
67.67
76.88
92.39
98.71
16.82
15.41
50.65
45.14
34.38
30.51
28.48
18.14
17.81
15.41
16.74
16.74
15.08
27.29
15.59
17.79
DPIIEDYH
ATLLDVIQSGVENL
ATLLDVIQSGVENLD
ASVHIKLPK
LIDDHFLFKEGDR
LIDDHFLFKEGDR
ATLLDVIQSGVENLDS
GVGIYAPDA
EVQQKLIDDHFLFK
EVQQKLIDDHFLFK
APLFDPIIEDYH
LRVAPEE
EVQQKLIDDHFLFKE
GDR
DCFMVV
HQGVMVGMGQK
FAGDDAPR
AVFPSIVGR
GFAGDDAPR
AGFAGDDAPR
AGFAGDDAPR
AGFAGDDAPRA
DCFMVV
DCFMVV
DCFMVV
SKMTSGTTIQIVG
IANVTVSNF
DDLTVTNPK
DMYMEFCKDFPIVSIE
DPFD
0.0000000.1
Amidated (Cterm)
0.000400.1
0.000400.1
0.000400.1
0.000400.1
0.040400000000000000
00.0
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
Amidated (Cterm);
Oxidation (M)
2 Oxidation (M)
453
gi|225714196|gb|ACO12944.1|
gi|225714196|gb|ACO12944.1|
gi|225714196|gb|ACO12944.1|
gi|148613137|gb|ABQ96193.1|
gi|321467477|gb|EFX78467.1|
gi|290562041|gb|ADD38417.1|
gi|290562041|gb|ADD38417.1|
gi|148613137|gb|ABQ96193.1|
gi|148613141|gb|ABQ96195.1|
gi|148613141|gb|ABQ96195.1|
gi|148613143|gb|ABQ96196.1|
gi|148613143|gb|ABQ96196.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
gi|551380|gb|AAB31477.1|
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
arginine kinase, AK [Penaeus
japonicus=shrimps, tail muscle, Peptide, 355 aa]
anti-lipopolysaccharide factor isoform 4
[Farfantepenaeus paulensis]
anti-lipopolysaccharide factor isoform 4
[Farfantepenaeus paulensis]
anti-lipopolysaccharide factor isoform 3
[Farfantepenaeus paulensis]
anti-lipopolysaccharide factor isoform 3
[Farfantepenaeus paulensis]
anti-lipopolysaccharide factor isoform 1
[Farfantepenaeus paulensis]
anti-lipopolysaccharide factor isoform 1
[Farfantepenaeus paulensis]
ABC protein, subfamily ABCH [Daphnia pulex]
14-3-3 protein zeta [Lepeophtheirus salmonis]
14-3-3 protein zeta [Lepeophtheirus salmonis]
Calcitonin gene-related peptide type 1 receptor
precursor [Lepeophtheirus salmonis]
Calcitonin gene-related peptide type 1 receptor
precursor [Lepeophtheirus salmonis]
Calcitonin gene-related peptide type 1 receptor
precursor [Lepeophtheirus salmonis]
15
15
15
35
22
28
28
35
35
35
35
35
454
454
454
454
454
454
454
454
454
454
15.41
15.6
16.74
15.56
22.09
27.78
15.55
35.13
15.56
35.13
15.56
35.13
16.22
17.12
19.78
21.06
21.23
22.98
28.5
37.36
38.24
38.36
SKFMCP
AENSCPDFP
SKFMCP
IRGEA
LLSLHGLSTL
LLETNMT
EAFDEAINDL
CPGWTPIRGEA
IRGEA
CPGWTPIRGEA
IRGEA
CPGWTPIRGEA
QTDKHPNKDF
ATLLDVIQSGVENLDS
GVGIYAPDA
MGLTEFQAVK
QTDKHPNKDF
ASVHIKLP
SVHIKLPK
FDPIIEDYH
GVENLDSGVGIYAPD
AEAY
GVENLDSGVGIYAPD
A
ATLLDVIQSGVENLDS
GVG
Oxidation (M)
Oxidation (M)
Amidated (Cterm)
Oxidation (M)
Oxidation (M)
Gln->pyro-Glu
(N-term Q)
Gln->pyro-Glu
(N-term Q)
0.000400.0
0.000000000.1
0.000400.0
0.0000040.0
2.0000000000.0
0.4000000000.0
2.0000000000.0
454
Proteins found in O. rusticus, PEAKS
Brain 1
Accession
gi|380003174
14-3-3 protein [Scylla paramamosain]
gi|321452467
ABC protein_ subfamily ABCC [Daphnia pulex]
gi|321467478
ABC protein_ subfamily ABCH [Daphnia pulex]
gi|395863637
antimicrobial peptide type 2 precursor [Pandalopsis japonica]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|551380
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|254832574
beta-actin [Penaeus monodon]
gi|211947526
beta-actin [Procambarus clarkii]
gi|211947526
beta-actin [Procambarus clarkii]
gi|225713794
Carboxypeptidase A2 precursor [Lepeophtheirus salmonis]
gi|121078585
caspase [Litopenaeus vannamei]
gi|296933602
caspase [Marsupenaeus japonicus]
gi|296933420
caspase [Marsupenaeus japonicus]
gi|99032198
Chain A_ 1.2a Resolution Structure Of A Crayfish Trypsin Complexed With A Peptide Inhibitor_ Sgti
gi|99032198
Chain A_ 1.2a Resolution Structure Of A Crayfish Trypsin Complexed With A Peptide Inhibitor_ Sgti
gi|402715413
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
gi|402715413
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
gi|402715413
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
gi|402715413
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Peptide
YYEILNSPDRACHLA
AVIVTFLAHIG
GGPPGTPDSGIPGP(-.98)
RPFGSGGR
APLFDPIIED
ATLLDVIQSGVENL
ATLLDVIQSGVENLDSGVG
DVIQSGVENLDSGVG
FAPLFDPIIED
FAPLFDPIIEDY
LFAPLFDPIIED
LIDDHFLFK
AGFAGDDAPR
AGFAGDDAPRAVF
AGFAGDDAPRAVFPS
AGFAGDDAPRAVFPSIVG
APLNPK
DDAPRAVFP
DDAPRAVFPSIVG
DEAQSKRGILT
DEAQSKRGILT
EAPLNPKANR
FAGDDAPR
FAGDDAPRAVFPS
FAGDDAPRAVFPSIVG
GDDAPRAVFPSIVG
HQGVM(+15.99)VGM(+15.99)GQ
LKYPIEHGIIT
LPHAILR
LTEAPLNPK
RGILTLK
TEAPLNPK
TEAPLNPK
TEAPLNPKANR
DSGDGVSHTVPIYEG
VSHTVPIYEG
PCEVIIAN
KNVPWGR
IGSALIR
IGSALLR
AGELDMSVNEGS(-.98)
IVGGTDATLGEFPYQL
AGAAELGIPLYR
DEGGFAPNIL
DEGGFAPNILN
DEGGFAPNILN
-10lgP
17.15
17.4
15.7
16.51
26.43
44.98
49.85
34.61
44.46
54.83
36.67
22.77
46.67
46.07
26.71
46.8
21.07
20.56
41.73
46.25
39.49
23.04
41.27
31.45
58.25
27.61
48.2
18.58
24.76
45.91
22.58
39.45
39.27
61.29
51.78
23.66
20.87
16.48
15.54
15.54
18.21
73.7
76.09
16.96
41.61
27.46
455
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|118175954
gi|290562063
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
corticotropin-releasing hormone binding protein [Tigriopus japonicus]
Geranylgeranyl transferase type-2 subunit beta [Lepeophtheirus salmonis]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
DEGGFAPNILNNK
EVILPVP
EVILPVPAF
EVILPVPAF
M(+15.99)GTEVYHHLK
NVINGGSHAGNK
NVINGGSHAGNKLA
QAKRSEGGPH(-.98)
WLAERQLPSGG(-.98)
DGPSAKDWR
GGRGAAQNIIPS
GGRGAAQNIIPSS
GGRGAAQNIIPSSTG
GPSAKDWR
GPSAKDWR
TVDGPSAKDWR
TVDGPSAKDWR
TVDGPSAKDWR
AM(+15.99)PYDNPIPGYK
FVPRTVM(+15.99)IGGK
GGKAAP
ILPRHLQI
LPAPHEP
LPAPHEP
LPRHLQ
VLYPNDNFFEGK
VVHLDQL
AERLSNFLPAVDEL
AETWNPRDHTDK
AINRLLYK
ALHNQAHRVLG
APHTTYK
ATRDPAF
ATRDPAFFR
DADEAVANGAELPH
DAERLSNFLPAVDEL
DEAVANGAELPH
DEAVANGAELPHK
DGFAPHTTY
DGFAPHTTYK
DGFAPHTTYKYG
DPAFFR
DTDVAHQQQAINR
DTDVAHQQQAINR
DTDVAHQQQAINRL
DTDVAHQQQAINRLL
DTDVAHQQQAINRLL
DTDVAHQQQAINRLLY
73.62
35.26
50.48
44.17
23.16
18.32
56.12
18.99
31.64
27.14
22.05
41.19
33.54
53.36
52.28
42.98
42.54
29.25
18.94
26.52
19.78
25.52
43
37.13
18.34
25.07
18.72
30.5
48.86
17.05
67.96
33.87
20.3
46
58.33
65.39
46.27
74.58
24.61
73.44
48.27
38.95
93.48
76.15
74.36
108.77
53.35
43.99
456
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
DTDVAHQQQAINRLLYK
E(-18.01)VTPHLF
EAAETWNPR
EAAETWNPRDHTDK
ETWNPRDHTD
ETWNPRDHTDK
EVTPHLF
EVTPHLFTNSE
EVTPHLFTNSEVIDQ(-.98)
FAPHTTYK
FNM(+15.99)PPGVM(+15.99)EHFET
FNMPPGVMEHFET
FPFDRPIPD
GAELPHKESR
GEFPARPDNK
GEFPARPDNK
GFAPHTTYK
GFPFDRPIPD
GFPFDRPIPD
KFNM(+15.99)PPGVM(+15.99)
KFNM(+15.99)PPGVM(+15.99)EHFET
LLLPKGK
LPPLYEVTPHLF
LSNFLPAVDEL
M(+15.99)PPGVM(+15.99)EHFET
M(+15.99)TQTPGNFK
NEHGIDILGDIIE
NEHGIDILGDIIE
NEHGIDILGDIIES
NEHGIDILGDIIESS
NEHGIDILGDIIESS
NQAHRVL
NQAHRVLG
PARPDNKEF
PHTTYK
SSESSVAIPDR
SSESSVAIPDRVSFPQ
SSESSVAIPDRVSFPQL
TDINNEHGIDILG
TWNPRDHTDK
VFKVQNQHGQVVK
VLGAQSDPK
VM(+15.99)EHFETATR
VQNQHGQVVKIFHH
VSFPQLIHDA
VSFPQLIHDADEAVAN
VTPHLFTNS
VVLPPLYEVTPH
112.14
39.28
18.98
19.97
34.77
59.17
31.81
41.42
60.72
38.74
54.78
39.48
27.7
45.08
49.92
39.16
35.48
42.62
27.8
21.79
51.53
28.64
55.88
41.53
51.21
47.15
44.78
19.14
22.47
51.97
40.19
18
19.87
15.8
39.13
28.08
42.62
67.26
15.09
38.52
79.99
18.75
68.94
83.88
27.7
51.4
49.69
22.55
457
gi|290462393
gi|225710178
gi|225717830
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|33087166
gi|387811963
gi|114386730
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|133754273
gi|326579691
gi|326579693
gi|290562011
gi|290562255
gi|310743902
gi|17223042
gi|321458499
gi|321458458
gi|321479308
gi|225709352
gi|74785595
gi|257096767
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hepatopancreas kazal-type proteinase inhibitor [Penaeus monodon]
hormone receptor-like 97a [Daphnia magna]
hyasin [Hyas araneus]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
inhibitor of apoptosis protein [Penaeus monodon]
JHE-like carboxylesterase 1 [Pandalopsis japonica]
JHE-like carboxylesterase 2 [Pandalopsis japonica]
Peptide deformylase_ mitochondrial [Lepeophtheirus salmonis]
Peptidyl-tRNA hydrolase 2_ mitochondrial [Lepeophtheirus salmonis]
phosphoenolpyruvate carboxykinase [Marsupenaeus japonicus]
putative antimicrobial peptide [Litopenaeus setiferus]
putative DH31 receptor [Daphnia pulex]
putative diuretic hormone 44 receptor [Daphnia pulex]
putative PDF receptor variant 2 [Daphnia pulex]
Pyrazinamidase/nicotinamidase [Caligus rogercresseyi]
RecName: Full=Armadillidin; Flags: Precursor
RecName: Full=Succinate dehydrogenase assembly factor 2_ mitochondrial; Short=SDH assembly
factor 2; AltName: Full=Succinate dehydrogenase subunit 5_ mitochondrial; Flags: Precursor
Serine/threonine-protein phosphatase 2A regulatory subunit B [Lepeophtheirus salmonis]
Signal recognition particle 54 kDa protein [Caligus rogercresseyi]
takeout precursor [Caligus clemensi]
VDEIPLE
NNALSSLSKA(-.98)
RLM(+15.99)PVGIPE
WAHHQLTAR
WNPRDHTD
YAAKM(+15.99)TQT
YDAERLSNFLPAVDEL
YGGEFPARPD
YGGEFPARPDNK
DPSFFR
DPSFFRLH
LHNTAHIM(+15.99)LG
LPNRFLLP
LPNRFLLPK
NTAHIM(+15.99)LGR
RVDDERIF
TAHIM(+15.99)LGR
TRDPSFFR
QEITVAYP(-.98)
APLEIK
PRPYRP
APVDIKE
E(-18.01)HAEVI
EAPVDIK
EPITTP
KPSEVQP
LKVTAPQAPT
M(+15.99)LKNNVRVNEIWSMFRAGE(-.98)
Q(-17.03)SLEGLK
QSAHVECRL
RETKAQSPI
RVWTPPP(-.98)
TATPTPKID(-.98)
VTVTENLQRADQEDV
EALKPPSSGPP(-.98)
LGAVLMGMN(-.98)
VTANEGAALT
QTLPPYE(-.98)
VVGIGPGP
KAKVIM(+15.99)HDPFA(-.98)
PGGFPGGRP(-.98)
LPAVVT
VIAGPT
M(+15.99)IKAPLD(-.98)
PAGENGEEVVPPI
GFHSGGS(-.98)
IPPYEERKNE(-.98)
29.95
18.62
22.39
20.81
24.91
18.87
77.39
43.87
81.07
31.57
42.05
18.95
22.48
22.83
59.21
22.21
18.37
43.85
15.05
15.66
15.94
19.72
15.16
23.49
16.28
27.9
17.81
21.09
15.45
15.9
20.16
15.02
18.61
15.33
16.06
18.04
18.01
26.39
27.2
16.53
16.18
28.11
15.97
30.86
15.68
16.15
19.27
458
Brain 2
gi|304360632
gi|225908473
gi|321465377
gi|321465377
Accession
gi|225710814
gi|66774602
gi|321460334
gi|321468387
gi|321473806
gi|381216270
gi|77168456
gi|155964257
gi|114386732
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|450
gi|450
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|211947526
gi|211947526
gi|225714196
tumor necrosis factor receptor-associated factor 6 [Litopenaeus vannamei]
ubiquitin carboxyl-terminal esterase L3 [Scylla paramamosain]
vitellogenin fused with superoxide dismutase [Daphnia pulex]
vitellogenin fused with superoxide dismutase [Daphnia pulex]
Description
14-3-3 protein beta/alpha [Caligus rogercresseyi]
14-3-3-like protein [Penaeus monodon]
ABC protein_ subfamily ABCH [Daphnia pulex]
ABC protein_ subfamily ABCH [Daphnia pulex]
ABC protein_ subfamily ABCH [Daphnia pulex]
androgenic hormone_ partial [Oniscus asellus]
antimicrobial peptide PEN2-4 [Litopenaeus vannamei]
anti-microbial Scy2 precursor [Scylla serrata]
arasin-1 [Hyas araneus]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
AVLLPKKTEKK
AVLLPKKTEKK
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Procambarus clarkii]
beta-actin [Procambarus clarkii]
Calcitonin gene-related peptide type 1 receptor precursor [Lepeophtheirus salmonis]
TVDLMYIEH(-.98)
VRWLPLESNPAVM(+15.99)NK
E(-18.01)AEPKLL
EAVEAEP
Peptide
ISSIGQKT
ASNPESKVFYLK
PSIPVEF(-.98)
LLDRNLVAGVKM(+15.01)
ILVLGHEP(-.98)
KVDLMFPLLE(-.98)
TGPIPRPPP
YTAVMS
SPGRPR
ATLLDVIQSGVENLDSGVG
ATLLDVIQSGVENL
FAPLFDPIIEDY
LFAPLFDPIIED
ASVHIKLP
APLFDPIIED
AVLLPKKTEKK
AVLLPK
HQGVMVGMGQK
EAPLNPKANR
AGFAGDDAPR
AGFAGDDAPR
AGFAGDDAPR
AGFAGDDAPR
APLNPKANR
DEAQSKRGILT
TEAPLNPK
TEAPLNPK
LTEAPLNPK
FAGDDAPRAVFP
VVDNGSGMVK
FAGDDAPR
FAGDDAPR
AGFAGDDAPRAVFPS
GFALPHAILR
M(+15.99)GQKDAYVGDEAQSK
DDAPRAVFPS
E(-18.01)APLNPK
AGFAGDDAPRA
HQGVM(+15.99)VGM(+15.99)GQK
RGILTLK
VSHTVPIYEG
SGDGVSHTVPIYEG
VSDEYL
21.05
26.28
16.35
20.71
-10lgP
15.47
36.19
18.57
17.84
16.31
17.23
19.56
16.05
17.59
54.02
46.63
42.15
38.21
30.29
15.02
42
29.3
67.73
53.1
50.84
46.1
39.57
34.42
50.68
48.45
43.4
34.95
41.25
39.6
36.92
36.42
19.02
35.69
28.58
28.07
27.31
23.32
19.93
19.3
16
48.29
15.74
19.06
459
gi|225714196
gi|225717916
gi|225708792
gi|290562677
gi|296933280
gi|296933378
gi|296933480
gi|296933780
gi|296933780
gi|99032198
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|402715413
gi|238244
gi|121615296
gi|210076885
gi|254351320
gi|162945363
gi|45385712
gi|193878327
gi|448
gi|154936860
gi|133754271
gi|133754271
gi|8698612
gi|225709118
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|62701385
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743896
gi|310743898
gi|318104933
gi|21914372
gi|21914372
gi|21914372
gi|21914372
Calcitonin gene-related peptide type 1 receptor precursor [Lepeophtheirus salmonis]
Carboxypeptidase B [Caligus clemensi]
Carboxypeptidase B [Caligus rogercresseyi]
Carboxypeptidase B [Lepeophtheirus salmonis]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
Chain A_ 1.2a Resolution Structure Of A Crayfish Trypsin Complexed With A Peptide Inhibitor_ Sgti
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
chymotrypsin Pm1 {EC 3.4.4.5} [Penaeus monodon=shrimps_ midgut_ Peptide Partial_ 31 aa]
crustin [Litopenaeus schmitti]
crustin [Paralithodes camtschaticus]
crustin antimicrobial peptide [Portunus trituberculatus]
crustin antimicrobial peptide [Scylla paramamosain]
crustin I [Litopenaeus vannamei]
crustin-2 [Eriocheir sinensis]
DLPKVDTALK
ecdysone receptor [Marsupenaeus japonicus]
effector caspase [Penaeus monodon]
effector caspase [Penaeus monodon]
eyestalk peptide [Jasus edwardsii]
GDP-fucose protein O-fucosyltransferase 1 precursor [Caligus rogercresseyi]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
glycogen synthase [Marsupenaeus japonicus]
glycogen synthase [Marsupenaeus japonicus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
LDPLRE(-.98)
IHTFM(+15.99)DQI
TPLM(+15.99)SLTLH(-.98)
LKLLRIEKAGSN(-.98)
PTGIKDCLF
ISAIQNKFEMTVE
IIGFFVGP
LPKTRYP
KYLEVVPP(-.98)
GTDATLGEFPYQ
DEGGFAPNILNNK
DEGGFAPNILNNK
NVINGGSHAGNKL
DEGGFAPNILN
DEGGFAPNIL
PIVSIEDPFDQ
NAVKNVN
GVEAVPHSWPYQAALFII
TPVGTKLLD
NDPLTPAQVA(-.98)
E(-18.01)ASLVLPYSGL(-.98)
KHHVCKT
TPVGTKILD
FSGGPP(-.98)
DLPKVDTALK
VM(+15.99)IGKLLNVLT
DALVVVFMSHGEK
TGLGERN
TPARQR
SFSAFVK
TVDGPSAKDWR
TVDGPSAKDWR
TVDGPSAKDWR
DGPSAKDWR
GVNLEKYSK
GGRGAAQNIIPSSTG
NNVVNTMRL
LPAPHEP
RDIFKDF
PAPHEP
SPNNFNLK
TNGITPR
LTTAINNIR
SELINSLLGLP(-.98)
VQNQHGQVVKIFHH
DTDVAHQQQAINR
DTDVAHQQQAINR
DTDVAHQQQAINR
17.81
20.09
17.4
15.41
20.15
15.4
16.37
19.69
15.78
28.75
65.54
50.53
58.27
40.25
28.56
27.02
17.47
18.85
21.6
28.82
20.29
16.78
21.6
18.6
43.41
17.69
22.38
19.82
18.55
23.58
53.22
22.69
15.78
43.88
32.54
23.81
34.42
31.48
30.63
24.93
19.61
17.06
15.96
21.08
80.87
79.45
66.87
56.91
460
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
DTDVAHQQQAINRLLY
YGGEFPARPDNK
KEAAETWNPRDHTDK
KEAAETWNPRDHTDK
EAAETWNPRDHTDK
EAAETWNPRDHTDK
DTDVAHQQQAINRL
YGGEFPARPDNKEF
LIHDADEAVANGAELPHK
AETWNPRDHTDK
AETWNPRDHTDK
DTDVAHQQQAINRLL
DTDVAHQQQAINRLL
ETWNPRDHTDK
VMEHFETATR
MPPGVMEHFET
LPPLYEVTPHLFTNS
VFKVQNQHGQVVK
GGEFPARPDNK
DGFAPHTTYK
VMEHFET
YGGEFPARPD
EVTPHLFTNSEVID
DPAFFR
EVTPHLFTNSE
FKVQNQHGQVVKIFHH
GEFPARPDNK
E(-18.01)VTPHLF
DPAFFRLH
FPQLIHDADEAVAN
GEFPARPDNKEF
APHTTYK
EVTPHLFTNS
NQHGQVVKIFHH
EVTPHLF
NEHGIDILGDIIES
GFAPHTTYK
DVAHQQQAINR
KVQNQHGQVVK
PHTTYK
AINRLLYK
RPIPDLR
LLLPKGK
AERLSNFLPAVDEL
ATRDPAFFRL
EVTPHLFTNSEVID(-.98)
SSESSVAIPDR
GFPFDRPIPDL
74.51
71.86
63.47
25.67
61.5
25.3
60.89
59.35
59.18
58.71
29.82
57.66
51.5
54.09
49.97
47.76
47.66
45.97
45.48
44
43.59
43.51
43.32
42.93
42.3
42.05
42.03
41.03
40.06
38.17
38.01
37.96
37.6
35.47
34.54
33.93
33.14
32.68
32.62
31.86
30.46
30.26
29.87
29.6
28.2
27.87
27.64
27.58
461
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|249511
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|133754273
gi|46398237
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
inhibitor of apoptosis protein [Penaeus monodon]
intestinal trypsin 5 precursor [Lepeophtheirus salmonis]
YGGEFPARP
IDRKGELF
EVTPHLFTNSEVIDQ
VTPHLFT
TRDPAFFR
E(-18.01)VTPHLFT
VM(+15.99)EHFET
LLVDVLLH
DGFAPHTTY
SSESSVAIPDRVSFPQ
EVTPHLFTNSEVIDQ(-.98)
VSFPQLIHDADEAVAN
DAERLSNFLPAVDEL
E(-18.01)VTPHLFTNS
FPFDRPIPD
FPFDRP
FPARPDN
DPAFFRLHK
PAFFRLH
ATRDPAFF
RLLLPK
EVTPHL
PARPDNK
EAAETWNPR
QYPDKRPHGYPLDR
NTAHIM(+15.99)LGR
DPSFFR
YMDNIFR
LPPGVLEHFET
RDPSFFR
LPPGVLEHF
SALGLPNR
ALELDRF
EVIHEAYKA(-.98)
ADVAPSIAPLE(-.98)
TPTPVLSWQKDGV(-.98)
DPNPLP(-.98)
TVEVVGPRPT
QADVAPSIAP(-.98)
DVTSSSL
KLVSQGKT(-.98)
TATPTPKID
KDIPEREPVELE(-.98)
KPKDKPEP(-.98)
IPGVKCFVK(-.98)
EGLEKIRQLE(-.98)
TFGSSDFVV
IVGGTEVSP(-.98)
27.13
26.99
26.92
26.82
26.53
26.42
25.47
24.57
24.55
23.74
23.61
23.22
22.5
21.74
19.92
19.36
19.27
17.99
17.91
17.91
17.87
16.83
16.16
15.53
48.22
44.69
37.07
34.67
31.61
31.04
30.33
29.22
22.46
18.74
29.33
23.03
23.02
21.65
17.83
17.21
17.11
16.66
15.41
15.37
15.35
15.15
15.49
18.23
462
Eyestalk
1
14-3-3 protein epsilon [Lepeophtheirus salmonis]
ABC protein_ subfamily ABCH [Daphnia pulex]
ABC protein_ subfamily ABCH [Daphnia pulex]
Abl interactor 2 [Lepeophtheirus salmonis]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
astacidin 2 [Pacifastacus leniusculus]
gi|290562958
gi|321467477
gi|321460334
gi|290462835
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|111118804
gi|290462393
gi|225710178
gi|225908473
gi|290561503
gi|148589360
gi|148589360
gi|95113635
gi|399936386
Accession
gi|257096767
putative antimicrobial peptide [Litopenaeus vannamei]
putative G-protein coupled receptor_ orphan [Daphnia pulex]
putative UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase [Mesocyclops edax]
RecName: Full=NAD-dependent protein deacylase; AltName: Full=Regulatory protein SIR2 homolog 5;
Flags: Precursor
RecName: Full=Succinate dehydrogenase assembly factor 2_ mitochondrial; Short=SDH assembly
factor 2; AltName: Full=Succinate dehydrogenase subunit 5_ mitochondrial; Flags: Precursor
Serine/threonine-protein phosphatase 2A regulatory subunit B [Lepeophtheirus salmonis]
Signal recognition particle 54 kDa protein [Caligus rogercresseyi]
ubiquitin carboxyl-terminal esterase L3 [Scylla paramamosain]
Ubiquitin carboxyl-terminal hydrolase isozyme L3 [Lepeophtheirus salmonis]
unnamed protein product [Cypridina noctiluca]
unnamed protein product [Cypridina noctiluca]
vitellogenin fused with superoxide dismutase [Daphnia magna]
voltage-gated calcium channel beta subunit transcript variant 3 [Scylla paramamosain]
Description
gi|17223030
gi|321477804
gi|157812760
gi|387935383
NKASEDKLNLIKN
VYLRYVSY
EEARQAGTI
PTQRPPSPP(-.98)
SVHIKLPK
SVHIKLPK
SVHIKLP
RMGLTEFQAVK
RMGLTEFQAV
RM(+15.99)GLTEFQAVK
RM(+15.99)GLTEFQAVK
RM(+15.99)GLTEFQAV
Q(-17.03)TDKHPNKDFG
Q(-17.03)TDKHPNKDF
Q(-17.03)TDKHPNKDF
LIDDHFLFK
LIDDHFLF
LIDDHFL
LFDPIIEDYH
IDDHFLFK
GTYFPLTGMSK
GTYFPL
FAPLFDPIIE
DDHFLFK
ATLLDVIQSGVENLDSGVGIYAPDAEAY
ATLLDVIQSGVENLDSGVGIYAPDA
ATLLDVIQSGVENLDSGVG
ATLLDVIQSGVENLD
ATLLDVIQSGVENL
APLFDPIIEDYH
VLVALM(+15.99)AAVP
KYMDLCRKIQR(-.98)
KGLFKGGDMS(-.98)
VRWLPLESNPAVM
GPINRGTT
NFKQLL
TVIEFFKLIVIDI
KNILSEKLNE(-.98)
PPLPPP(-.98)
Peptide
RARLLYQ
IKAVILCGL
AALLDRL
E(-18.01)FAAAH(-.98)
IAEFEHRM(+15.99)T
18.26
15.43
23.76
15.3
42.39
36.22
29.98
53.64
54.99
50.89
31.03
20.99
16.91
37.52
19.5
35.09
24.89
29.53
48.07
34.98
41.97
24.68
29.65
34.49
22.72
100.64
84.56
70.28
57.79
87.59
15.66
19.44
15.32
51.97
27.29
19.16
16.65
18.12
20.52
-10lgP
18.35
16.57
27.98
20.83
47.29
463
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|321478601
gi|290561028
gi|304569876
gi|296933604
gi|296933318
gi|321474388
gi|159163115
gi|402715413
gi|121615293
gi|262385516
gi|254351320
gi|229459067
gi|258618631
gi|46240814
gi|46240814
gi|46240814
gi|321470051
gi|154936860
gi|226934556
gi|226934556
gi|284981903
gi|290462553
gi|62701385
gi|310743896
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|249511
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
Btk family kinase at 29A-like protein [Daphnia pulex]
Carboxypeptidase B [Lepeophtheirus salmonis]
caspase [Eriocheir sinensis]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
CG13830-PA-like protein [Daphnia pulex]
Chain A_ Solution Structure Of The [t8a]-Penaeidin-3
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
crustin [Farfantepenaeus paulensis]
crustin 1 [Procambarus clarkii]
crustin antimicrobial peptide [Portunus trituberculatus]
crustin Pm4 antimicrobial peptide [Penaeus monodon]
crustin-like antimicrobial peptide [Fenneropenaeus indicus]
crustin-like peptide type 3 [Marsupenaeus japonicus]
crustin-like peptide type 3 [Marsupenaeus japonicus]
crustin-like peptide type 3 [Marsupenaeus japonicus]
dissatisfaction-like protein [Daphnia pulex]
ecdysone receptor [Marsupenaeus japonicus]
ecdysteroid receptor isoform [Neomysis integer]
ecdysteroid receptor isoform [Neomysis integer]
fortilin binding protein [Penaeus monodon]
Geranylgeranyl transferase type-2 subunit beta [Lepeophtheirus salmonis]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
TEAPLNPK
DAYVGDEAQSK
AVFPSIVGR
AGFAGDDAPRAVFPSIVG
Q(-17.03)TVKVVEAVDPA
LKEQDISAEVI
Q(-17.03)CREVREM(+15.99)A
PEYQFYSSFTCW(-.98)
FFFTTHADT
DSVNQLDD
PLPGGPI
FAGKNFR
FGGGGVGGGF
PCLSLN(-.98)
E(-18.01)ASLVLPYSGL(-.98)
GGGGVNGGGL
PGHGGIAPGFE(-.98)
GVQGGGVGGV
GGGFGGGFGGPQGGG
GFGGVQGGGV
FGGGGGGGGGG(-.98)
FGDLLREDQ
YFDNEPY(-.98)
AHQLRVSSLD
PPCPYSK(-.98)
IQIAAIYDSM(+15.99)
TVDGPSAKDWR
PAADLSEQIST
YGGEFPARPDNK
YGGEFPARPDN
VSFPQLIHDADEAVAN
VSFPQLIHDA
VFKVQNQHGQVVK
VFKVQNQHGQVV
PMGFPFDRPIPDL
PGNFKM(+15.99)
LLLPKGK
GGEFPARPDNK
GFPFDRPIPDL
FPFDRPIPDL
EAAETWNPR
DTDVAHQQQAINR
DTDVAHQQQAINR
DTDVAHQQQAIN
DGFAPHTTYK
DEAVANGAELPH
AETWNPR
KELGETFNPQGD(-.98)
34.19
43.65
36.52
56.23
16.05
25.52
21.14
26.58
25.89
17.5
20.04
30.99
19.22
17.17
16.55
19.96
18.27
20.65
25.24
17.65
43.96
19.16
21.28
15.8
15.08
16.91
33.18
20.19
85.4
65
26.76
30.25
65.63
59.42
32.78
22.94
32.41
16.15
43.5
33.55
43.12
105.32
83.35
56.92
49.7
22.65
19.87
16.03
464
Eyestalk
2
14-3-3 protein beta/alpha [Caligus rogercresseyi]
ABC protein_ subfamily ABCH [Daphnia pulex]
antimicrobial peptide [Penaeus monodon]
antimicrobial peptide type 1 precursor [Pandalopsis japonica]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
gi|225710814
gi|321473806
gi|317383206
gi|395863635
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|321465377
Accession
gi|14285771
gi|225710178
gi|2655270
gi|158266420
gi|251354
gi|257096767
gi|6015045
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hormone receptor-like 97a [Daphnia magna]
hormone receptor-like 97a [Daphnia magna]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
phantom [Marsupenaeus japonicus]
putative DH31 receptor [Daphnia pulex]
putative PDF receptor variant 2 [Daphnia pulex]
Pyrazinamidase/nicotinamidase [Caligus clemensi]
RecName: Full=Nuclear hormone receptor E75; AltName: Full=Nuclear receptor subfamily 1 group D
member 3
RecName: Full=Nuclear hormone receptor E75; AltName: Full=Nuclear receptor subfamily 1 group D
member 3
RecName: Full=Succinate dehydrogenase assembly factor 2_ mitochondrial; Short=SDH assembly
factor 2; AltName: Full=Succinate dehydrogenase subunit 5_ mitochondrial; Flags: Precursor
RecName: Full=Superoxide dismutase [Mn]_ mitochondrial; Flags: Precursor
Signal recognition particle 54 kDa protein [Caligus rogercresseyi]
small heat shock/alpha-crystallin protein precursor [Artemia franciscana]
stylicine 2 [Litopenaeus stylirostris]
vacuolar H(+)-ATPase proteolipid subunit homolog [Nephrops norvegicus_ hepatopancreas_ Peptide
Partial_ 151 aa]
vitellogenin fused with superoxide dismutase [Daphnia pulex]
Description
gi|249511
gi|387811963
gi|387811963
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|224471277
gi|321458499
gi|321479308
gi|225717724
gi|6015045
E(-18.01)KVEGELR
IYLLPPFLST
VPGVGVPGVGGG
SPRSPIP(-.98)
APLFDPIIEDYH
ASVHIKLP
ASVHIKLP
ASVHIKLPK
ATLLDVIQSGVENL
ATLLDVIQSGVENLD
ATLLDVIQSGVENLDSGVG
ATLLDVIQSGVENLDSGVGIYAPDA
DPIIEDYH
AM(+15.99)GPWFTKLMEF(-.98)
Peptide
DPLEATTGLVP
AGGAGVM(+15.99)EM(+15.99)
TPARTTRSGG(-.98)
RLPGWEPP
TLYQGFVHM(+15.99)GAGL
IPPYEERKNE(-.98)
KAKILAAMQS
DPSFFR
QPPTSQDTNNNQP
NEASAPAENTYHP(-.98)
VRTTEDDEGMYS(-.98)
SPPTAPLEVRPT
RFKTSYDF
QTELLDDVTKPMPVVA
PIEEKF
PDGVTEPVKP(-.98)
KPRFLT
KLKKKPEPKI
IIEPEPEPEPELIILRPVE
E(-18.01)VSDKIPELA(-.98)
E(-18.01)PQATGKFR
ALTGPSQIM(+15.99)E
AAPIEEKF
KHPAFMPFQ
YFLVSNYF(-.98)
M(+15.99)IKAPLD(-.98)
TAKDSM(+15.99)ELGF(-.98)
KMTEHTAA
16.55
15.13
15.37
15.69
87.17
36.88
19.31
59.85
46.56
65.5
40.82
55.77
52.02
15.8
-10lgP
27.45
19.76
16.8
20.47
16.19
33.16
18.84
33.81
17.24
18.99
16.84
16.04
15.77
20.6
20.65
17.16
19.01
17.53
70.08
21.22
22.14
16.21
41.66
18.61
15.64
46.42
20.75
18.17
465
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|551380
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|254832574
gi|290561028
gi|296933458
gi|296933666
gi|296933806
gi|385048424
gi|157881028
gi|402715413
gi|238245
gi|118175954
gi|46240814
gi|46240814
gi|73426824
gi|448
gi|448
gi|290784917
gi|402169217
gi|402169217
gi|402169217
gi|290562063
gi|290562063
gi|62701385
gi|62701385
gi|310743896
gi|321458864
gi|321458864
gi|21914372
gi|21914372
gi|21914372
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
arginine kinase_ AK [Penaeus japonicus=shrimps_ tail muscle_ Peptide_ 355 aa]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
beta-actin [Penaeus monodon]
Carboxypeptidase B [Lepeophtheirus salmonis]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
caspase [Marsupenaeus japonicus]
caspase 8_ partial [Daphnia pulex]
Chain A_ Nmr Structure Of The Synthetic Penaeidin 4
Chain A_ X-Ray Structure And Catalytic Mechanism Of Lobster Enolase
chymotrypsin Pm2 {EC 3.4.4.5} [Penaeus monodon=shrimps_ midgut_ Peptide Partial_ 31 aa]
corticotropin-releasing hormone binding protein [Tigriopus japonicus]
crustin-like peptide type 3 [Marsupenaeus japonicus]
crustin-like peptide type 3 [Marsupenaeus japonicus]
crustin-like protein fc-2 [Fenneropenaeus chinensis]
DLPKVDTALK
DLPKVDTALK
duplex-specific nuclease [Chionoecetes opilio]
ecdysone phosphate phosphatase [Daphnia magna]
ecdysone phosphate phosphatase [Daphnia magna]
ecdysone phosphate phosphatase [Daphnia magna]
Geranylgeranyl transferase type-2 subunit beta [Lepeophtheirus salmonis]
Geranylgeranyl transferase type-2 subunit beta [Lepeophtheirus salmonis]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glyceraldehyde 3-phosphate dehydrogenase [Marsupenaeus japonicus]
glycogen phosphorylase [Marsupenaeus japonicus]
GPCR-like protein_ family B [Daphnia pulex]
GPCR-like protein_ family B [Daphnia pulex]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
EVQQKLIDDHFLFK
FDPIIEDYH
GVENLDSGVGIYAPDA
GVENLDSGVGIYAPDAEAY
HIKLPK
IDDHFLFK
LIDDHFLF
LIDDHFLF
LIDDHFLF
M(+15.99)GLTEFQAVK
Q(-17.03)TDKHPNKDF
Q(-17.03)TDKHPNKDF
SVHIKLPK
TEAQYKEM(+15.99)QQ(-.98)
AGFAGDDAPR
AGFAGDDAPRAVFPSIVG
AVFPSIVGR
FAGDDAPR
GFAGDDAPR
HQGVMVGMGQK
LKEQDISAEVI
LLLATTVALHN(-.98)
QLPPATTVALHN
TPKKKAKLFF(-.98)
LKQEDF
RPIFIRP(-.98)
AGAAELGIPLYR
VGGVEAVPGV
TTLLFPANFE(-.98)
GFGGVQGGGV
GPQGGGFGG
VPGVGGGFVP
DLPKVDTALK
PKVDTALK
IVRIAAGATV(-.98)
ILGETQAGLLGQAL(-.98)
LIGHASTLD
LVGGLP
ILYTLSA
IQIAAIYDSM
DGPSAKDWR
TVDGPSAKDWR
Q(-17.03)GKKWVDTQIVFAM(+15.99)
ITVKYLRLT
M(+15.99)FISILLNSILVIVF
AVTHSDLTPH(-.98)
DGFAPHTTY
DGFAPHTTYK
75.5
35.31
23.53
59.8
17.38
23.88
32.51
23.62
23.35
39.45
26.92
26.12
46.58
15.12
30.29
23.98
47.75
40.04
27.39
77.03
36.42
15.76
23.1
15.24
18.01
18.66
60.66
18.98
18.57
25.64
20.44
16.91
22.66
40.14
16.97
15.52
21.13
20.05
21.65
16.17
21.15
23.8
16.79
15.5
21.24
15.03
26.96
60.13
466
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|21914372
gi|249511
gi|249511
gi|387811965
gi|387811965
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|15425681
gi|46398233
gi|326579693
gi|298684192
gi|225717516
gi|76786564
gi|119655552
gi|356483023
gi|321458458
gi|321458458
gi|321479308
gi|225717724
gi|74785595
gi|74785595
gi|74836523
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin [Pacifastacus leniusculus]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hemocyanin subunit c [Panulirus interruptus=spiny lobster_ Peptide_ 661 aa]
hormone receptor-like 97b [Daphnia magna]
hormone receptor-like 97b [Daphnia magna]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
I-connectin [Procambarus clarkii]
intestinal trypsin 3 precursor [Lepeophtheirus salmonis]
JHE-like carboxylesterase 2 [Pandalopsis japonica]
Kazal-type serine proteinase inhibitor 2 [Procambarus clarkii]
Mitogen-activated protein kinase kinase kinase 9 [Caligus clemensi]
PEN2-1 [Litopenaeus vannamei]
peritrophin 3 precursor [Penaeus monodon]
putative crustin-like antimicrobial peptide [Nephrops norvegicus]
putative diuretic hormone 44 receptor [Daphnia pulex]
putative diuretic hormone 44 receptor [Daphnia pulex]
putative PDF receptor variant 2 [Daphnia pulex]
Pyrazinamidase/nicotinamidase [Caligus clemensi]
RecName: Full=Armadillidin; Flags: Precursor
RecName: Full=Armadillidin; Flags: Precursor
RecName: Full=Scygonadin; Flags: Precursor
DLKELESR
DPAFFR
DTDVAHQQQAIN
DTDVAHQQQAINR
EAAETWNPR
FNMPPGVMEHFE
GEFPARPDNK
GFPFDRPIPDL
MGFPFDRPIPDL
MTQTPGNFK
PMGFPFDRPIPDL
RPMGFPFDRPIPDL
TATRDPAFF
VAHGYIINADGT
VSFPQLIHDA
VVLPPL
YGGEFPARPD
YGGEFPARPDNK
YGGEFPARPDNKEF
DPSFFR
YGGYFPSRPDN
IESIVTKYM
TSDCGTEQDAC(-.98)
AAPIEEKF
APIEEKF
E(-18.01)AKATVTEELK
ISQDTVQV
KNHITLKVTAP
KPIETSSRINRTH
LEGRIEPV(-.98)
LEPLKPLK
LSKANLYFDT
QTHQKEKEFL
SEEVTIKKPEEKI
ISALFFLV
LEAPVIST
PVGRLCGRY(-.98)
KIRPDVLIDWSIQMAR
IPRPPPI
YSKLREK
TVVTVALGSGH
LIPLLGLTYVL(-.98)
WGIPLPVVIVWAVV(-.98)
LLCWPPT
LKLAENSIIIHK
GFNRGGGF
GGFNRGGGF
KLMPKIVSAII
29.2
27.99
60.99
110.22
33.46
58.8
33.94
18.01
45.57
24.34
36.67
31.38
27.93
20.01
50.3
43.54
53.47
89.07
45.81
29.56
40.91
17.34
15.2
27.55
15.21
15.81
17.21
17
16.67
22.96
16.77
23.25
15.01
19.36
22.28
25.83
15.65
16.66
16.79
15.32
23.22
24.66
17.89
15.84
19.03
33.19
18.48
20.32
467
gi|71040960
gi|71040960
gi|290462393
gi|290462393
gi|2655270
gi|2655270
gi|304360632
gi|304360632
gi|95113635
gi|95113635
gi|225711432
RXRd nuclear hormone receptor [Gecarcinus lateralis]
RXRd nuclear hormone receptor [Gecarcinus lateralis]
Serine/threonine-protein phosphatase 2A regulatory subunit B [Lepeophtheirus salmonis]
Serine/threonine-protein phosphatase 2A regulatory subunit B [Lepeophtheirus salmonis]
small heat shock/alpha-crystallin protein precursor [Artemia franciscana]
small heat shock/alpha-crystallin protein precursor [Artemia franciscana]
tumor necrosis factor receptor-associated factor 6 [Litopenaeus vannamei]
tumor necrosis factor receptor-associated factor 6 [Litopenaeus vannamei]
vitellogenin fused with superoxide dismutase [Daphnia magna]
vitellogenin fused with superoxide dismutase [Daphnia magna]
Zinc finger MYND domain-containing protein 19 [Caligus rogercresseyi]
ASEVPCANP(-.98)
TM(+15.99)GMKREAA(-.98)
VDEIPLEEQSQ
VDEIPLEEQSQR
APAVGRIEGGTTGTT
FGFGGFG(-.98)
KGGADSGIE
LSNLAKRIEEVDAL(-.98)
DPSSKYTSDKIN
E(-18.01)TVAIGKEE(-.98)
RVLLRYYN(-.98)
19.7
18.39
21.82
15.48
15.35
19.95
18.38
15.89
19.86
18.43
16.68
468
Protein Identifications from O. rusticus, Hypothetical Proteins from Daphnia pulex genome, Mascot
Protein
Mascot
Brain 1
Protein Accession #
Protein Description
Score
hypothetical protein
gi|321455882|gb|EFX67003.1|
DAPPUDRAFT_189558 [Daphnia pulex]
hypothetical protein
gi|321455882|gb|EFX67003.1|
DAPPUDRAFT_189558 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477436|gb|EFX88395.1|
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321477714|gb|EFX88672.1|
DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein
gi|321473719|gb|EFX84686.1|
DAPPUDRAFT_209520 [Daphnia pulex]
20.13
15.95
27.85
24.99
23.24
21.18
20.67
20.2
18.97
18.04
17.24
15.66
15.42
15.12
22.31
19.3
18.82
17.44
17.31
16.5
15.8
29.53
20
20
25
25
25
25
25
25
25
25
25
25
25
25
19
19
19
19
19
19
19
30
Peptide
Mascot
Score
EAQDIL
KLMAATRGTKLE
DLKKIEDLGAILEE
KALDE
YVKKIEPGLS
QTATILN
QGDNVEALIKK
QEIEN
ICQQKLMQSYA
VISLI
RYPDGIHYG
PLNAPF
RIVLDR
LKISSEMR
REELQR
RIVLDR
LETPPAFPI
EQILEASPLLE
RIVLDR
CNQTWRNEDPEA
VA
QINKANQY
EMMSSTGRTTPSS
LT
Peptide Sequence
Glu->pyro-Glu (N-term E)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term);
Oxidation (M)
Amidated (C-term)
Glu->pyro-Glu (N-term E)
Amidated (C-term)
Glu->pyro-Glu (N-term E);
2 Oxidation (M)
Peptide Variable
Modifications
3.000000.0
0.0000000.1
0.00000000000.1
0.00000.1
0.00000040000.0
0.000000.1
0.000000.1
0.00000040.1
0.000000.1
3.00000000000.0
0.000000.1
3.044000000000000.
0
Position of Variable
Modifications
469
gi|321469429|gb|EFX80409.1|
gi|321469429|gb|EFX80409.1|
gi|321469652|gb|EFX80631.1|
gi|321469652|gb|EFX80631.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
gi|321473719|gb|EFX84686.1|
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303899 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303899 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_304111 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_304111 [Daphnia pulex]
45
45
23
23
27
27
27
27
27
27
27
27
27
27
30
30
30
30
30
30
30
30
30
30
30.63
46.25
18.33
22.89
15.28
15.36
15.92
16.23
18.07
18.59
21.25
24.04
29.08
31.18
15.2
15.66
15.74
17.21
18.56
20.12
20.57
22.23
25.61
27.99
APPRPPPP
APPRPPPP
KEIAA
GAPVD
QPGDFILAIN
DMNAKNASG
QSQPIEI
EAVRTLLDLGASP
AGNLE
GADREALNYA
DDATILENHK
KTMTGMKD
EVEIPP
SLQLIR
QEAFLNNDDL
GQMME
EQLMAAGHFA
EKLVAAGEIH
KDNLNAGEAAR
LELDTYSGLINEM
KDLPQV
QDIDQMLD
QEIEI
QGLEN
Amidated (C-term)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
Oxidation (M)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
0.00000.1
0.0000000000.1
0.040000000.0
0.0000000000000.1
0.0000000000.1
0.0000000000.1
0.00000400.0
0.00000.1
0.0004000000.0
0.0000000000004.0
0.00000000.1
0.00000.1
470
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321460846|gb|EFX71884.1|
gi|321460846|gb|EFX71884.1|
gi|321460846|gb|EFX71884.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321461797|gb|EFX72825.1|
gi|321461797|gb|EFX72825.1|
gi|321461797|gb|EFX72825.1|
gi|321461797|gb|EFX72825.1|
gi|321463228|gb|EFX74245.1|
gi|321463228|gb|EFX74245.1|
gi|321476821|gb|EFX87781.1|
gi|321476821|gb|EFX87781.1|
gi|321469429|gb|EFX80409.1|
gi|321469429|gb|EFX80409.1|
gi|321469429|gb|EFX80409.1|
gi|321469429|gb|EFX80409.1|
hypothetical protein
DAPPUDRAFT_304111 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_304111 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_304111 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_304111 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_306505 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_306505 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_307391 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_307391 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308055 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308055 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308055 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308055 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
23
23
23
23
45
45
45
20
20
20
20
20
18
18
18
18
56
56
24
24
45
45
45
45
19.28
20.2
23.18
30.75
15.67
30.63
46.25
15.04
15.84
16.06
18.9
20.42
15.69
17.31
17.63
25.61
19.94
55.65
17.99
23.52
15.3
15.48
19.36
23.6
LARDPDE
SIIGGQDAEAFT
AVLLLL
EQIIGQELKSGGLE
KI
KPVPPPKPKN
APPRPPPP
APPRPPPP
LTPLPP
KNIESGAIRS
EVPKIT
LQYNSLLE
ETTTAAPSSETKP
LDIRK
KADLE
SIPPPPPPL
KEIEI
AVVDDSQK
ASNPESKVFYLK
AVQKLMVHNW
SSRAPPR
LSGRINFQIQYQLS
F
APDLLI
SSRAPPR
SSRAPPR
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term); Glu>pyro-Glu (N-term E)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
0.0000000.1
0.000000.1
0.000000.1
0.00000000.1
3.0000000000000.1
0.000000000.1
0.00000000.1
0.0000000000.1
0.000000000000000.
1
0.000000.1
471
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321473952|gb|EFX84918.1|
gi|321473952|gb|EFX84918.1|
gi|321473952|gb|EFX84918.1|
gi|321474567|gb|EFX85532.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_314077 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_314509 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_314509 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_314509 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
22
22
22
22
22
22
22
22
22
22
22
22
22
27
27
27
23
23
23
23
23
23
23
23
16.62
16.82
17.54
17.82
18.36
18.43
18.8
19.48
20.27
21.33
22.97
24.23
30.75
15.73
18.78
27.39
22.89
15
15.27
15.37
15.68
16.03
16.17
17.93
SLSIR
TIAFKLESPQ
QPLTTLLE
KPRDSSN
ISAETTCSDF
KEPSPTKL
HLDLP
PAMQAEGIPI
PIRCSLVVGAKLG
DQLEPF
PTTCALPLH
ADVVQMAS
GATAGEPKNA
AVLLLL
WLDLEKPMNRQ
QLELSLTDP
KDPEGVPLN
GAPVD
RLVLNNFR
QPCPATW
NPIVA
QTMYTANV
GDVAVVQP
QPATGAIIT
AGEPMP
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term);
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Oxidation (M)
0.0000000000.1
0.00000000.1
0.0000000.1
0.0000000000.1
0.00000400.1
0.0000000000.1
0.000000.1
0.00000000000.1
0.000000000.1
0.00000.1
0.00000000.1
0.000040.0
472
gi|321462130|gb|EFX73155.1|
gi|321462130|gb|EFX73155.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_325390 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_325390 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
24
24
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
27.32
30.73
15.07
15.24
15.28
15.28
15.42
16
16.29
16.41
16.58
17.1
17.45
17.93
18.66
20.14
21.01
21.57
22.49
15.26
15.28
15.58
15.75
16.58
IAQEGVDMF
ISKLLGL
QPTMQPTMQ
LIATSPPP
QLIMIR
MQPMMQPMMQP
MMQPMMQ
MISPP
GRLLVL
QPMMQPV
QPMVQPI
LSDIDMDGRL
QTGLFP
PAMQPTMQPTMQ
PAMQPM
ETADLSSLSP
EQEERERK
PMVQPIMSSNLP
RGILVQ
LIATSPPPT
KAVCTHAVLEL
EESSSPILGQGEIV
DN
LEVLAKIPV
QAVVSL
WIQEVAISVQR
TGLLNKLMH
Oxidation (M)
Oxidation (M)
4 Oxidation (M)
Amidated (C-term); Gln>pyro-Glu (N-term Q);
Oxidation (M)
Oxidation (M)
Oxidation (M)
Oxidation (M)
2 Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Gln->pyro-Glu (N-term Q)
Oxidation (M)
0.000000040.0
0.000000040.0
2.000400.1
0.400440000000000
040.0
0.0004000.0
0.0040000.0
0.0000040000.0
0.000000.1
0.000000400000000
004.0
0.00000000.1
0.000000.1
0.000000000000000
0.1
2.000000.0
0.000000040.0
473
Brain 2
gi|321468653|gb|EFX79637.1|
gi|321468653|gb|EFX79637.1|
gi|321468653|gb|EFX79637.1|
gi|321468653|gb|EFX79637.1|
gi|321468030|gb|EFX79017.1|
gi|321473999|gb|EFX84965.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321470602|gb|EFX81578.1|
gi|321470602|gb|EFX81578.1|
gi|321470602|gb|EFX81578.1|
gi|321470602|gb|EFX81578.1|
gi|321470602|gb|EFX81578.1|
gi|321470602|gb|EFX81578.1|
Protein Accession #
gi|321454896|gb|EFX66047.1|
gi|321454896|gb|EFX66047.1|
gi|321462130|gb|EFX73155.1|
gi|321462130|gb|EFX73155.1|
gi|321462130|gb|EFX73155.1|
Protein Description
hypothetical protein
DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_194294 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_225308 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_30197 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_30197 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_30197 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_30197 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_325390 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_325390 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_325390 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_332604 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_332604 [Daphnia pulex]
Protein
Mascot
Score
34
34
34
34
21
24
24
24
24
24
24
24
35
35
35
35
35
35
19
19
24
24
24
15.14
17.68
17.92
33.66
20.63
23.97
15.22
15.33
16.08
16.1
17.26
24.4
15.08
15.59
17.2
17.4
18.04
34.9
18.94
Peptide
Mascot
Score
22.27
16.08
18.48
24.37
RQPPM
ELLKPGHGRDR
LAGGGGGELALG
RQPPM
EQILE
ESEAVAIRDSLAA
NSGFVKA
PFRVGIFCVLDQL
DR
ISSEMRAKLE
KASPGSIGKLKIS
EPMSRIEARK
AEKEPLE
ELKPVEKK
CNQTWRNEDPEA
VA
PPPPSRV
KELKPVEK
DPPNRS
APPPPPPPPPPP
APPPPPPPPPPP
Peptide Sequence
ISPSSCTNK
AGGLNK
SLKGSPEAVLNSL
N
GLGDIEGLIDK
GAGGLGNLMGM
GG
Oxidation (M)
Amidated (C-term); Glu>pyro-Glu (N-term E)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term);
Oxidation (M)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Peptide Variable
Modifications
Amidated (C-term)
Amidated (C-term); 2
Oxidation (M)
0.00004.0
3.00000000000.1
0.00004.0
0.000000000000000
00000.1
0.000000000000000.
1
0.0000400000.1
0.0040000000.0
0.0000000.1
0.00000000.1
0.0000000.1
0.000000.1
Position of Variable
Modifications
0.00000000000000.1
0.0000000040400.1
474
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321477347|gb|EFX88306.1|
gi|321477347|gb|EFX88306.1|
gi|321463228|gb|EFX74245.1|
gi|321463228|gb|EFX74245.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_307391 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_307391 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311561 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311561 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
25
25
25
25
25
25
25
31
31
31
31
31
31
31
31
17
17
106
106
29
29
29
29
29
18.17
20.53
21.83
21.94
26.23
28.05
30.52
15.28
15.41
15.59
15.59
16.47
19.78
24.21
30.52
15.13
17.44
64.1
66.45
15.39
17.19
18.97
21.29
29.33
ISAETTCSDF
ISAETTCSDFIRLL
T
HPSCPPIAL
LILPNDL
PAMQAEGIPI
LILPNDL
AVLLLL
IKGAWS
GDVAVVQP
PPPPDIL
EVVTPN
NDNAPRF
GLMPT
QPDGPAIK
AVLLLL
MPPKSPDLIE
DTESPTPTNSAVTI
D
ASNPESKVFYLK
ASNPESKVFYLK
EDVVAFKETP
QKTSAEL
KDFNP
QVKSAKNEEPRVS
VTVNSTPLIPDPD
GGALIDY
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term); Glu>pyro-Glu (N-term E)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
0.0000000.1
0.0000000000.1
0.0000000.1
0.000000.1
0.000000.1
0.00400.0
0.000000.1
0.0000000000.1
0.000000000000000.
1
3.0000000000.1
0.00000.1
0.000000000000000
00000000000.1
0.0000000.1
475
Eyestalk
1
gi|321462615|gb|EFX73637.1|
gi|321462615|gb|EFX73637.1|
gi|321462615|gb|EFX73637.1|
gi|321462615|gb|EFX73637.1|
gi|321462615|gb|EFX73637.1|
gi|321462615|gb|EFX73637.1|
Protein Accession #
gi|321470570|gb|EFX81546.1|
gi|321470570|gb|EFX81546.1|
gi|321470570|gb|EFX81546.1|
gi|321464766|gb|EFX75772.1|
gi|321464766|gb|EFX75772.1|
gi|321464766|gb|EFX75772.1|
gi|321465056|gb|EFX76060.1|
gi|321465056|gb|EFX76060.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
gi|321470568|gb|EFX81544.1|
Protein Description
hypothetical protein
DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_323032 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_323032 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_323032 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_50104 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_50104 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_50104 [Daphnia pulex]
Protein
Mascot
Score
31
31
31
31
31
31
35
35
35
26
26
26
19
19
15
15
15
15
25
25
25
25
25
17.4
16.31
16.39
20.36
20.53
21.64
31.06
Peptide
Mascot
Score
18.04
34.9
19.74
22.84
26.41
15.63
18.56
15.12
19.42
22.47
23.09
15.33
15.6
16.47
16.54
16.85
QIAEMQA
SSPVPVAD
EFPGPTESAPLPPP
P
ENEELEVLLAF
GAGAGLIQSGKQ
KIVRQNKL
Peptide Sequence
DPPNRS
APPPPPPPPPPP
APPPPPPPPPPP
GQVSISN
GQVSISN
QGSVDSVVING
GQVVEIL
KILMLLDE
QPMVQPI
QPLVQPMMQPLM
QPMMQP
EGIQELPENVS
LMATNMQS
SHSEELAD
EQLTTLLRM
QPIRCSLVVGAK
ISAETTCSDFIRLL
T
TQPLTTLLEE
Amidated (C-term)
Glu->pyro-Glu (N-term E)
Amidated (C-term)
Amidated (C-term)
Peptide Variable
Modifications
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Gln->pyro-Glu (N-term Q)
Oxidation (M)
Amidated (C-term);
Oxidation (M)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term); 2
Oxidation (M)
Amidated (C-term)
Amidated (C-term); Gln>pyro-Glu (N-term Q)
0.0000000.1
0.00000000.1
3.000000000000000.
0
0.00000000000.1
Position of Variable
Modifications
0.000000.1
0.0000000.1
0.0000000.1
2.00000000000.0
0.00040000.1
0.0040000.0
0.000000000004000
000.0
0.00000000000.1
0.04000400.1
0.00000000.1
2.000000000000.1
0.0000000000.1
476
Eyestalk
2
Protein Accession #
gi|321474231|gb|EFX85196.1|
gi|321457271|gb|EFX68361.1|
gi|321457271|gb|EFX68361.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321478348|gb|EFX89305.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321475268|gb|EFX86231.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321476878|gb|EFX87838.1|
gi|321475051|gb|EFX86015.1|
gi|321475051|gb|EFX86015.1|
gi|321475051|gb|EFX86015.1|
gi|321475051|gb|EFX86015.1|
gi|321475051|gb|EFX86015.1|
Protein Description
hypothetical protein
DAPPUDRAFT_301481 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_300356 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_301481 [Daphnia pulex]
Protein
Mascot
Score
26
15
15
31
31
31
29
29
29
29
15
15
15
15
15
15
15
20
20
20
20
20
25.83
Peptide
Mascot
Score
15.12
15.78
15.97
30.63
31.05
16.67
16.87
17.18
28.59
15.12
15.14
15.7
15.8
16.33
19.47
21.01
15.11
17.78
18.65
20.15
23.83
Peptide Sequence
SSPDDNASPR
SLFQSQNG
SELEASYAKGLAK
LASKLLKTTGELQ
GPHGTVTAGWSA
VAMKM
LPGMTVVCTEA
SLQLIR
EMVVTT
TSSSPGTRV
KSILL
TRVAA
YSLIGGNTQ
MDSLRPIRRETRD
L
LSGDVAVVQPLD
NSVTLRLGDMDQ
FITMAREPT
CLRKE
QLGGLP
SCLRKE
DPFVGPLVSVML
AVRNFVCR
KNVEVTVRVCN
HLGSG
KDLVAKH
Peptide Variable
Modifications
2 Oxidation (M)
Glu->pyro-Glu (N-term E)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Position of Variable
Modifications
0.000000000000000
00000000000000000
00000000404.0
3.000000.0
0.00000.1
0.000000000400.0
0.00000000000.1
0.0000000.1
477
gi|321478626|gb|EFX89583.1|
gi|321478626|gb|EFX89583.1|
gi|321478626|gb|EFX89583.1|
gi|321478626|gb|EFX89583.1|
gi|321478626|gb|EFX89583.1|
gi|321478626|gb|EFX89583.1|
gi|321478626|gb|EFX89583.1|
gi|321474528|gb|EFX85493.1|
gi|321474528|gb|EFX85493.1|
gi|321470892|gb|EFX81866.1|
gi|321470448|gb|EFX81424.1|
gi|321470448|gb|EFX81424.1|
gi|321469585|gb|EFX80565.1|
gi|321469585|gb|EFX80565.1|
gi|321469585|gb|EFX80565.1|
gi|321464134|gb|EFX75144.1|
gi|321464134|gb|EFX75144.1|
gi|321464134|gb|EFX75144.1|
gi|321474937|gb|EFX85901.1|
gi|321474937|gb|EFX85901.1|
gi|321474937|gb|EFX85901.1|
gi|321470625|gb|EFX81600.1|
gi|321470625|gb|EFX81600.1|
gi|321470625|gb|EFX81600.1|
hypothetical protein
DAPPUDRAFT_314171 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_314171 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_49826 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_49826 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_49826 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_45411 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_45411 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_45411 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_323685 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_323685 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_323685 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_318494 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_318494 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_318494 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_317013 [Daphnia pulex]
15
15
15
15
15
15
15
23
23
44
34
34
27
27
27
25
25
25
22
22
22
20
20
20
15.01
15.41
15.47
15.94
16.01
16.74
17.09
18.36
23.34
44.45
20
34.23
15.05
20.87
26.86
15.66
21.37
25.29
17.15
18.64
22.48
16.16
18.78
20.19
KSDLAWVAGKR
QHPMCP
SVLLMAI
KGFTCMPA
TTLRPTTT
QHPMCP
LTGRIADI
LLTGLL
FSPPAST
GAELLSFSMALTI
VCVWILTGHWLL
M
EGIQELPENVS
QPAMQPTMQ
PMMAPPLSM
LVQQPKG
LPVPMMAPP
DLIKACTT
LNLEDLITLP
LIRIDRA
ALSMIKTNFIVPLS
S
LRDSIIFPSSEH
LVCNLGMAD
WMMGTVLRT
GDLEKKAAMEQK
RTLTGDEANGH
GDLEKKAAMEQK
RTLTGDEANGH
Oxidation (M)
Oxidation (M)
Amidated (C-term)
Amidated (C-term);
Oxidation (M)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
Amidated (C-term); 2
Oxidation (M)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term);
Oxidation (M)
Amidated (C-term)
Oxidation (M)
Amidated (C-term)
2 Oxidation (M)
Amidated (C-term)
0.000400.0
0.000400.0
0.00000000.1
0.000000000000000
00000000004.1
0.00000000000.1
0.000400000.0
0.0000000.1
0.000044000.1
0.0000000000.1
0.0000000.1
0.000400000000000.
1
0.000000000000.1
0.000000400.0
0.044000000.0
0.000000000000000
00000000.1
0.000000000000000
00000000.1
478
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321477436|gb|EFX88395.1|
gi|321470311|gb|EFX81288.1|
gi|321470311|gb|EFX81288.1|
hypothetical protein
DAPPUDRAFT_242543 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_242543 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein
DAPPUDRAFT_191611 [Daphnia pulex]
26
26
26
26
26
26
26
26
26
17
17
15.27
15.62
15.83
16.73
17.17
18.15
18.34
21.52
26.35
15.24
17.36
QDSSNALSIF
IEEITAI
ILEASPLL
ILEASPLL
QQQQPN
GFLDKNRDT
LLSFHKGD
EELQRREE
IGRIS
AADLDHMPTMKE
TKYL
ELQEMNGMNLMG
PP
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term)
Amidated (C-term); Glu>pyro-Glu (N-term E)
0.0000000.1
0.000000.1
0.00000000.1
0.000000000000000
0.1
3.00000000000000.1
479
Protein Identifications from O. rusticus, Hypothetical Proteins from Daphnia pulex genome, PEAKS
Brain 1
Accession
gi|321472102
hypothetical protein DAPPUDRAFT_101007 [Daphnia pulex]
gi|321470602
hypothetical protein DAPPUDRAFT_102305 [Daphnia pulex]
gi|321466199
hypothetical protein DAPPUDRAFT_106344 [Daphnia pulex]
gi|321466199
hypothetical protein DAPPUDRAFT_106344 [Daphnia pulex]
gi|321464736
hypothetical protein DAPPUDRAFT_11648 [Daphnia pulex]
gi|321465242
hypothetical protein DAPPUDRAFT_188571 [Daphnia pulex]
gi|321477436
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
gi|321477436
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
gi|321477436
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
gi|321473723
hypothetical protein DAPPUDRAFT_194402 [Daphnia pulex]
gi|321472278
hypothetical protein DAPPUDRAFT_195011 [Daphnia pulex]
gi|321471029
hypothetical protein DAPPUDRAFT_195819 [Daphnia pulex]
gi|321467013
hypothetical protein DAPPUDRAFT_198141 [Daphnia pulex]
gi|321460747
hypothetical protein DAPPUDRAFT_201492 [Daphnia pulex]
gi|321477714
hypothetical protein DAPPUDRAFT_206384 [Daphnia pulex]
gi|321457437
hypothetical protein DAPPUDRAFT_301440 [Daphnia pulex]
gi|321478348
hypothetical protein DAPPUDRAFT_303153 [Daphnia pulex]
gi|321478348
hypothetical protein DAPPUDRAFT_303153 [Daphnia pulex]
gi|321478626
hypothetical protein DAPPUDRAFT_303213 [Daphnia pulex]
gi|321478626
hypothetical protein DAPPUDRAFT_303213 [Daphnia pulex]
gi|321468823
hypothetical protein DAPPUDRAFT_304375 [Daphnia pulex]
gi|321468823
hypothetical protein DAPPUDRAFT_304375 [Daphnia pulex]
gi|321468289
hypothetical protein DAPPUDRAFT_304927 [Daphnia pulex]
gi|321467722
hypothetical protein DAPPUDRAFT_305142 [Daphnia pulex]
gi|321477408
hypothetical protein DAPPUDRAFT_305633 [Daphnia pulex]
gi|321466003
hypothetical protein DAPPUDRAFT_305989 [Daphnia pulex]
gi|321464495
hypothetical protein DAPPUDRAFT_306711 [Daphnia pulex]
gi|321460846
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
gi|321460846
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
gi|321460846
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
gi|321478464
hypothetical protein DAPPUDRAFT_310476 [Daphnia pulex]
gi|321475865
hypothetical protein DAPPUDRAFT_312745 [Daphnia pulex]
gi|321475864
hypothetical protein DAPPUDRAFT_312746 [Daphnia pulex]
gi|321475051
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
gi|321475051
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
gi|321475051
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
gi|321474567
hypothetical protein DAPPUDRAFT_314077 [Daphnia pulex]
gi|321470892
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
gi|321470892
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
gi|321470892
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
gi|321470568
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
gi|321470568
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
Peptide
FSSVGPS(-.98)
SSPSLPPT
RQPPPPPP
TPSAAP
IPAVVT
AVLLLFPLNDKFESY
DPGSPTQPN(-.98)
NQPVISLIAKKPI
RSPNPPGP
ENLKPVYPCT
TQYTIGSST
VLVGSE
TFTCVAKNSAGF
PIPPLPDK
TM(+15.99)EALEDTWRN
TVPELNIT
NGEDLAKA
TDCGLLEGWLR
HDGRKKNM(+15.99)FDAPIE
SLGCNGSADQQTTV(-.98)
DLMSIGMGKPA
VGPSWGSPAA
ELWINGKLTDY(-.98)
KKWAEGDFK(-.98)
VPEPARSPSPP
PDSVAPLDM(-.98)
SIFDSIK
ADGEDAPPRP
APPRPPPP
APPRPPPP
E(-18.01)SIASEVDEVY(-.98)
QGVDLNR
KKANLIKLYLTL
HVFELN(-.98)
LTPPDQEIM(+15.99)D(-.98)
SIGGRG(-.98)
VVLSHGENGM(+15.99)IY(-.98)
GELIKHTENDH
TVLAKHDEGG(-.98)
VGNEPIVGM(+15.99)DN
HGPIFRGIGP(-.98)
LFQSGLL
-10lgP
19.92
19.24
17.22
15.65
28.11
15.48
16.14
18.18
20.33
21.42
18.61
16.79
16.46
19.27
20.59
21.02
15.26
18.73
17.38
21.17
18.03
17.02
18.97
15.05
19.77
19.89
18.44
17.68
45.54
27.73
25.22
16.14
28.07
15.35
19.34
15.29
18.04
15.5
23.93
20.57
17.66
17.8
480
Brain 2
gi|321470568
gi|321470448
gi|321470448
gi|321470448
gi|321469585
gi|321477671
gi|321477671
gi|321477671
gi|321477376
gi|321470910
gi|321462615
gi|321477575
gi|321474610
Accession
gi|321472102
gi|321470602
gi|321470602
gi|321470602
gi|321455473
gi|321477436
gi|321475531
gi|321473999
gi|321473723
gi|321473723
gi|321461543
gi|321473719
gi|321473719
gi|321461113
gi|321479055
gi|321461456
gi|321471172
gi|321471172
gi|321478348
gi|321478626
gi|321469744
gi|321469429
gi|321468905
gi|321477300
gi|321477408
gi|321466003
gi|321462341
gi|321461745
gi|321460934
gi|321460846
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_318494 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_4688 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_49564 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_95322 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_98778 [Daphnia pulex]
Description
hypothetical protein DAPPUDRAFT_101007 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_102305 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_116167 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_192770 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_194294 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_194402 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_194402 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_201000 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_216285 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_232322 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290667 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_302612 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_302612 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303875 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304111 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304348 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_305524 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_305633 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_305989 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_307720 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308094 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308626 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
QASSFASS(-.98)
LIATSPPP
LVNAASGSLT
TVVPGENHP
PPM(+15.99)ALGLPVP
ASALGDLI
LLEKPNEP(-.98)
TASVITLTSGPP
DAGIFSC
LSKIRQ(-.98)
EFLQDAEGLVTLGLM(-.98)
APAKDD(-.98)
PKLPEPP
Peptide
QPWRGPTDVT
APPPPPPPPPPP
APPPPPPPPPPP
QPDVRGAL
NDPIYTR
CNQTWRNEDPEAVA
TPLGNSDIL(-.98)
ESEAVAIRDSLAANSGFVKA(-.98)
NADKRIT
DGKPIPGSS(-.98)
PLEAIDLYN
DPTNLQSKLQ
WINEHMITASSED(-.98)
TAGIVGGLST
SIPSVK(-.98)
EVFLPLKNIK
RIVIKMAAD
RNSLLLAPMP
M(+15.99)EDRLQSQQCDMA
LLPGLK
TPLFLALVRGDIKTLF
PPPPKASAQLKAVQNA
VVSAEGKRIQ(-.98)
PATLKILGAH(-.98)
SPVITEPVIR(-.98)
VPIPDPM(+15.99)P
RNSPASSPRLRT
CRHNKSLSIVYK
KDELM(+15.99)ILMQSM(+15.99)
PPRPPPP
20.11
25.32
15.33
16.14
21.34
17.88
16.68
16.99
15.03
15.87
18.39
19.14
19.51
-10lgP
15.71
37.75
23.24
15.22
21.15
18.64
15.77
16.34
17.82
15.7
15.54
24.06
19.07
27.49
19.68
20.1
16.43
15.89
18.13
17.1
29.63
15.97
15.32
15.09
26.47
18.51
24.76
16.42
16.48
44.42
481
Eyestalk 1
gi|321460846
gi|321460846
gi|321460846
gi|321460846
gi|321460846
gi|321460261
gi|321468635
gi|321477347
gi|321476878
gi|321476878
gi|321476878
gi|321476878
gi|321475864
gi|321475051
gi|321475051
gi|321475051
gi|321474202
gi|321470892
gi|321470892
gi|321470892
gi|321470892
gi|321470568
gi|321470568
gi|321470568
gi|321470568
gi|321470448
gi|321470448
gi|321470448
gi|321470448
gi|321469372
gi|321466438
gi|321466438
gi|321465056
gi|321462130
gi|321458311
gi|321443655
gi|321477671
gi|321473455
gi|321470625
gi|321462615
gi|321460687
gi|321478212
gi|321474610
Accession
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308950 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_31061 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311561 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_312746 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313770 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_318481 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_321304 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_321304 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_325390 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_329158 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_345055 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_46980 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_49826 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_57919 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_59834 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_95253 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_98778 [Daphnia pulex]
PPRPPPP
APPRPPPP
TPGAVDKLN
VTAVQPGSPAA
PPTLDPR
SVVAVPHERIVG
GIYLLCLGERDI(-.98)
PAVPPRVSPSM(+15.99)
EGYGNLR
NNYVKNPVLFD(-.98)
PAWSSQLP
YAIYRTAGEILP(-.98)
LPVSNILPV(-.98)
PDSASVVQL
DLMSNRSKMLT
Q(-17.03)LESFT
PPGTLALGDGS(-.98)
VVIIPATT
PEQLYMVTGRCEI(-.98)
KTSLGM(+15.99)APT(-.98)
SLGMAPTT
VPPHGP
YTSSTSVVAPPP(-.98)
HVELPH
QLLLACLAVFT
M(+15.99)MQPLMQPMM(+15.99)
AGTPPSSMMGAPL
KGQTGLFP(-.98)
PHM(+15.99)SPYIRFCSRQ
DCPLTILGET
IFTMIPYT
LGVIPP
IEIDFR
ISKLLGL
VDILLSM(+15.99)GISRN(-.98)
GIQRLIISGIR(-.98)
KTANADPSAP
DM(+15.99)ISVIDM(+15.99)PPP(-.98)
PAVLSATT
QTLQGMKESGLK(-.98)
SLGAAAIEAT
IKGVFEGF
EPIVGA(-.98)
Peptide
26.36
40.76
21.66
15.79
15.31
15.4
22.32
18.55
22.15
16.47
16.35
15.87
16.97
21.79
20.49
16.83
18.02
31.31
17.6
16.8
15.66
25.21
21.56
21.26
19.77
25.75
19.63
17.61
15.51
17.8
28.74
19.5
18.09
24.57
19.57
20.31
17.16
18.96
18.41
18.74
19.16
17.25
17.78
-10lgP
482
gi|321472102
gi|321469722
gi|321466199
gi|321464736
gi|321477436
gi|321476638
gi|321471029
gi|321468642
gi|321467013
gi|321464553
gi|321461543
gi|321460747
gi|321460747
gi|321473719
gi|321468920
gi|321475130
gi|321472279
gi|321460227
gi|321461456
gi|321479333
gi|321458870
gi|321457271
gi|321471172
gi|321478626
gi|321478626
gi|321468905
gi|321468905
gi|321468289
gi|321464495
gi|321464495
gi|321464495
gi|321463123
gi|321461797
gi|321461745
gi|321461745
gi|321460934
gi|321460846
gi|321460846
gi|321460729
gi|321478464
gi|321476878
gi|321476878
gi|321476878
gi|321475051
hypothetical protein DAPPUDRAFT_101007 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_103233 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_106344 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_11648 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_192312 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_195819 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_197424 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_198141 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_199303 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_201000 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_201492 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_201492 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_224893 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_236642 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_239963 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290661 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290667 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_300115 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_300640 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_301481 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_302612 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303213 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304348 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304348 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304927 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_306711 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_306711 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_306711 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_307445 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308055 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308094 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308094 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308626 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308744 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_310476 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311854 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
QPIINSPHELEN(-.98)
E(-18.01)SAAAQVNGRP(-.98)
Q(-17.03)TTLAPQP(-.98)
ERHLPGY
PDAAQKRPAT
E(-18.01)ADIVNR
QGKMCVLDCSPN(-.98)
DSAEGGAGPTTPA
EIKKKPKVRK
DYNDMKDL
SGGAAGGP
VSEANQGSNP
NVAPAIQQQQ
IDAASSSHE(-.98)
APNEKPLQV
QQDDELCFCKH(-.98)
KAIFVDGGI
HGQHVLSVPLTL(-.98)
SLAVADM(+15.99)LM(+15.01)
DPLGAPSLS(-.98)
GPVPQGLV
E(-18.01)ELVKPLK
RPGAFAIY
TPGGIK
DKPTDEDIYNY(-.98)
GDM(+15.99)PEPKY(-.98)
EQLRDEVYCQ(-.98)
PARVKDIPLQDM(+15.01)
SIFDSIK
SIFDSIK
Q(-17.03)QPVANM(+15.99)TAS
TDIM(+15.99)ESKS(-.98)
PLNLILEPT
SYVSVARSF
DPGVAH(-.98)
VSPAGVVNQSD
NGHHPSQRDP
KGGPAEG
PQIGM(+15.99)VHP
KNGGDIQMIKPSAFK
PSPTPPHNMM(+15.01)
E(-18.01)VM(+15.99)AGDGRSQPR
DNVPVLND(-.98)
TQWIPTPEKD(-.98)
19.73
16.82
16.74
16.14
15.79
19.21
20.4
21.41
15.54
21.12
17.15
15.24
19.99
19.97
18.4
17.68
16.34
34
20.39
17.99
25.02
22.55
19.22
26.31
15.37
16.55
15.99
16.55
26.72
19.72
18.2
16.65
17.45
16.39
18.62
18.95
15.63
22.16
15.03
15.43
15.95
16.74
17
16.39
483
Eyestalk 2
gi|321475051
gi|321475051
gi|321474567
gi|321470892
gi|321470706
gi|321470568
gi|321470448
gi|321469478
gi|321465056
gi|321465056
gi|321465056
gi|321465056
gi|321477671
gi|321477671
gi|321470910
gi|321470625
gi|321453462
Accession
gi|321472102
gi|321472102
gi|321472102
gi|321466199
gi|321466199
gi|321464736
gi|321460217
gi|321455882
gi|321477436
gi|321477436
gi|321473999
gi|321477714
gi|321473719
gi|321473719
gi|321470311
gi|321470311
gi|321455555
gi|321460227
gi|321460227
gi|321460227
gi|321461456
gi|321458600
gi|321478348
gi|321478733
gi|321468905
gi|321468823
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_314077 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317013 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317361 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317679 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_318657 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_49564 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_49826 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_93775 [Daphnia pulex]
Description
hypothetical protein DAPPUDRAFT_101007 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_101007 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_101007 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_106344 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_106344 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_11648 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_15026 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_189558 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_191611 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_194294 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_206384 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_209520 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_242543 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_242543 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_262733 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290661 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290661 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290661 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_290667 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_300857 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303153 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_303168 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304348 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304375 [Daphnia pulex]
QSPVVQEVTSVLRE(-.98)
PAILSALM(+15.99)K
NIIAWISS
VLAKHDEGGN
PPPPGPPSF(-.98)
GVQGLDE(-.98)
LSFVAGEM(+15.99)VTII
PSSGGPGAA(-.98)
VLIAEPIEN(-.98)
SGGGTPTNPTP
LAGVALGGDIRYSGVTMLHPS
GGGSGGGSGG(-.98)
VM(+15.99)VTNVT
QM(+15.99)MM(+15.99)LDQSKT
ESPVPSLP
QTCSMALNM(+15.99)KT
KPIAWFDCNIH(-.98)
Peptide
LGLQNASLSTAT
LPQPVTIRF(-.98)
LPQPVTIRF(-.98)
KEAIAQER
QPPPPPPAPE(-.98)
RDAHNLVLCF(-.98)
IIQACRGNSP(-.98)
E(-18.01)RKTDVTY
AADLDHMPT
IAKKPIGVLHLLD(-.98)
QEMASNNSLV(-.98)
IVDPNRDGY(-.98)
LEEETRKAE(-.98)
LVAASLD
IVGPLSD(-.98)
VDNEGRKV(-.98)
LSRECPKPST
HGQHVLSVPLTL(-.98)
HGQHVLSVPLTL(-.98)
LDLSQTAITN(-.98)
LPKKKDGPCGSGSF(-.98)
FLPLSDT(-.98)
LLALVGGGAL(-.98)
LLTASGGSF(-.98)
TITAAHA
E(-18.01)KLTGTQQQQ
15.9
17.11
15.55
17.43
23.52
26.79
17.82
19.76
19.18
16.08
17.8
16.53
18.87
26.94
16.47
18.96
18.08
-10lgP
16.8
22.57
16.22
17.36
19.15
19.48
21.01
18.14
16.46
24.4
15.34
20.54
15.6
20.67
18.05
18.61
34.5
21.24
16.61
15.36
19.98
15.25
15.55
17.42
15.54
21.05
484
gi|321468823
gi|321468823
gi|321468823
gi|321468220
gi|321468220
gi|321477408
gi|321464495
gi|321462341
gi|321461745
gi|321461569
gi|321475268
gi|321460846
gi|321460846
gi|321460846
gi|321460729
gi|321478464
gi|321478573
gi|321468635
gi|321477347
gi|321475051
gi|321470706
gi|321470568
gi|321470568
gi|321465056
gi|321465056
gi|321464134
gi|321458311
gi|321454896
gi|321477671
gi|321477671
gi|321477671
gi|321474247
gi|321470910
gi|321470570
gi|321470570
gi|321468494
gi|321460687
hypothetical protein DAPPUDRAFT_304375 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304375 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304375 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304918 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_304918 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_305633 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_306711 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_307720 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308094 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308200 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308511 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308707 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_308744 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_310476 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_310476 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_31061 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_311561 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_313508 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317361 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_317519 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_322731 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_323685 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_329158 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_332604 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_41019 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_45861 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_49564 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_50104 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_50104 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_52222 [Daphnia pulex]
hypothetical protein DAPPUDRAFT_59834 [Daphnia pulex]
LSQSNGSNPR
QCISTVSGQQP(-.98)
SSPARPVV
GVQALQNTYG
HDLPAEYDEM(+15.99)LRVM(+15.01)
VPAQTQQHIA(-.98)
SIFDSIK
CNILKARA(-.98)
KAGCEPDEE(-.98)
ETAINALAKDVV
PTATASGPPSAT
IVIISHRAAA(-.98)
KNEKKKKK
LLGAVGGSIGGPGQAG(-.98)
M(+15.99)ARAIGT
HQM(+15.99)EMYIDMSPALKSF(-.98)
LLYTLSA
LAVSTSEVSLL(-.98)
SPTGSVASPG
SGGGGGLGV
LTERIGIFPI
LNKLMHNM(+15.99)GSP(-.98)
RLTITGR
AAAAAAPTPKKR(-.98)
AAAAAQSGHPSSL
LNLEDLITLP(-.98)
E(-18.01)IAATWIFD(-.98)
SVVVRSEKGRKC(-.98)
LPTLQGLATVAH(-.98)
LSADGSSFV(-.98)
VM(+15.99)VTNVT
LQM(+15.99)YAH(-.98)
E(-18.01)APSDTM(+15.99)SE(-.98)
ISPVEN(-.98)
LPPTPAQP
IGDKNHPKF
LGGPVD(-.98)
21.76
23.48
15.05
15.38
19.71
17.7
29.26
15.05
16.15
15.03
15.27
16.22
15.54
17.56
19.28
17.06
21.65
17.61
16.99
35.28
20.26
21.12
18.29
21.66
20.24
19.16
18.2
15.69
19.81
17.14
33.17
15.39
23.45
16.68
18.03
17.36
23.92
485

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