Biosolutions 9D - Applied Biosystems

Transcription

Biosystems
Solutions
European Edition Issue 10 Summer 2004
The Magazine of Integrated Science
Announcing
the Family of
Real-Time
PCR Platforms
from Applied Biosystems
Genome-wide
Expression Profiling
Determination of the Doping Agent
Tetrahydrogestrinone
by LC-APCI-MS/MS
FRIEND&FOE:
in Search of a More Tolerant Immune System
Biosystems Solutions
Contents
Welcome to Biosystems Solutions
Today, traditional laboratory research is being complemented with information-based science that will open up new horizons.
This will have a huge impact on our understanding of biology, medicine and information science.
We are pleased to release the latest issue of Biosystems Solutions, which includes a wealth of knowledge in an exciting and varied mix
of articles that reflect scientific research today. This will undoubtedly be a valuable source of information for your life science discovery.
We are sure you will enjoy Biosystems Solutions Issue 10, and as always your comments and suggestions are more than welcome.
Contact us at: [email protected]
Cover Graphic
Cells in the bloodstream with foreign bodies. Red blood cells (red) have a biconcave shape. Neutrophil white blood cells (blue, transparent) have a
three-lobed nucleus (pink). These cells are part of the immune response, and help to keep the blood free of foreign bodies (spiky). These represent
allergens, hormones or viruses.
Key
The icons below help you to easily see which articles contain information related to Proteomics, Informatics, LC/MS and Molecular Biology.
Proteomics
Informatics
LC/MS
Molecular Biology
cover story
05
FRIEND & FOE:
Imagine being able to modify the immune system to deter its troublesome responses, while leaving its diseasefighting capabilities intact. That’s what the Immune Tolerance Network (ITN), an international network of
basic and clinical researchers that specialise in a wide-range of immune-related diseases, hopes to accomplish.
applications
Successful connections
Efficient primary structure elucidation of disulfide-bridged peptides.....................................................................................................................
Determination of the doping agent tetrahydrogestrinone by LC-APCI-MS/MS
The designer steroid tetrahydrogestrinone (THG) was identified in urine samples of elite athletes.......................................................................
Characterisation of the proteome of synaptic proteins from rat brain using cICAT® Reagent-LC MS/MS
In combination with 2D-gel electrophoresis MS and MS/MS...............................................................................................................................
Peptidomics®
Differential analysis and inventory of human cerebrospinal fluid..........................................................................................................................
Determination of residues of chloramphenicol in milk and milk products using HPLC/MS/MS
In the European Union the use of chloramphenicol (CAP) as a veterinary drug for food producing animals is prohibited...................................
Applied Biosystems technology arms researchers in the battle against SARS
In February 2003, a mysterious illness resembling pneumonia began to spread rapidly through different parts of Asia.........................................
High-throughput gene expression system
For a liver carcinoma research project at CEPH, Paris...........................................................................................................................................
Proteomics Solution in a leading edge research environment
Scientists involved in protein expression analysis today have more and more to deal with.....................................................................................
Detecting a peptide biomarker for hypertension in plasma
Peptide quantitation using the 4000 Q TRAP™ System.........................................................................................................................................
02BiosystemsSolutions
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The Magazine of Integrated Science
applications
…continued
Genome-wide expression profiling with the Applied Biosystems Expression Array System
Expression profiling of over 30,000 genes in paired normal and breast tumour tissue...................................................................................................
Assessment of apoptosis and lead compound cytotoxicity
Using the Applied Biosystems 8200 Cellular Detection System.....................................................................................................................................
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product news
New BioTrekker™ Software v1.0
Offers a solution to your genotyping data management problems ................................................................................................................................
NanoMate® 100 System
For the QSTAR®, API 3000™, API 4000™ & 4000 Q TRAP™ Systems........................................................................................................................
New amine specific iTRAQ™ Reagents
Expand multiplexing and quantitation capabilities for Proteomic researchers................................................................................................................
Applied Biosystems LIMS for the Life Sciences
Transforms the way your laboratory operates.................................................................................................................................................................
Announcing the family of Real-Time PCR platforms from Applied Biosystems
Instruments to suit all requirements..............................................................................................................................................................................
SQL*LIMS® v5 Software
The best in LIMS just got better...................................................................................................................................................................................
Introducing Tempus™ Blood RNA Tubes
Stabilise RNA profiles in whole blood for 5 days!.........................................................................................................................................................
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promotions
Special Offers for GeneAmp® PCR Systems and AmpliTaq Gold® DNA Polymerase
Discounts available until 31 July 2004..........................................................................................................................................................................
Achieve better, faster synthesis and superior peptides
Take advantage of our Special Offer and try out the most versatile activator chemistry, HATU....................................................................................
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customer focus
German researcher wins Applied Biosystems award for excellence in mass spectrometry
In recognition of outstanding work in the area of protein analysis................................................................................................................................
BioQualification Service
Supporting the Qualification process in your laboratory with the appropriate documentation and agreements.............................................................
SM
High throughput genotyping using ABI PRISM® 7900HT Sequence Detection System
A partnership between a CNRS laboratory and the Applied Biosystems and Genoscreen companies............................................................................
Genomics Spotlight
Dr Raija L.P. Lindberg, Head of the Clinical Neuroimmunology Laboratory, University Hospital of Basel..................................................................
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bio-highlights
Research using the 8500 Affinity Chip Analyzer
Presented at two forthcoming conferences.....................................................................................................................................................................
Applied Biosystems asks >>> What are your Real-time PCR needs?
AB-ONLINE >>> News on demand for integrated science............................................................................................................................................
Narrowing in on your mutations
Applied Biosystems now offers two new tools to significantly ease the work and to shorten the time between hypothesis and result............................
LCMS food and environmental applications binder
Applied Biosystems Mass Spectrometry systems are helping scientists solve analytical problems....................................................................................
Customer Training Courses
The knowledge required to achieve the very best results from your research..................................................................................................................
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BiosystemsSolutions03
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Use less sample.
The new Applied Biosystems Expression Array System—the most
complete and most sensitive human genome expression analysis
system available. Thanks to advanced design and novel chemiluminescence
chemistry, now you can detect expression events at lower levels and on more genes than
ever before—using a minimal amount of sample. With one integrated workflow and a
single microarray built on today’s most complete dataset, you get unambiguous, high-quality results that drive increased research
productivity. And to accelerate your decision making even faster, the system comes complete with valuable functional
information and annotation, links to the Celera Discovery System™, plus off-the-shelf TaqMan® Gene Expression Assays matched
to the arrays. Want to see more? For information and free webinars visit: http://info.appliedbiosystems.com/microarray
iScience. Applied Biosystems provides the innovative
products, services, and knowledge resources that are
enabling new, integrated approaches to scientific discovery.
cover story
Friend & Foe:
Mark Springer, BioBeat® Online Magazine
(www.biobeat.com)
I
magine being able to modify the immune system to
deter its troublesome responses, while leaving
its disease-fighting capabilities intact. That’s what
the Immune Tolerance Network (ITN), an international
network of basic and clinical researchers that
specialise in a wide-range of immune-related diseases,
hopes to accomplish.
ITN has teamed with Applied Biosystems in a multi-year
research agreement to help elucidate the molecular
mechanisms underlying immune tolerance and provide a
basis for developing methods to induce such tolerance.
ITN works with Applied Biosystems instrument
technologies, biological content and services, operating in
concert to provide an Integrated Science (iScience)
solution for researchers studying gene expression.
A world-class contract research group equipped
with advanced high-throughput analysis platforms,
high-quality biological content, and industry-leading bioinformatics
Page 6
cover story
Superior alternative to immunosuppressive agents
The challenge for the ITN scientists is to develop, implement,
and assess strategies that enable the immune system to
become tolerant to specific antigens. “What we’re trying
to achieve is a therapeutic that will allow people to
eliminate the use of lifelong immunosuppressants,”
says Dr. Vicki Seyfert-Margolis, the ITN Executive Director
of Tolerance Assays. “That may mean you take a short-term
therapy to induce immune tolerance, and then you’re drug-free
for the rest of your life.” If successful, it would transform
the practice of organ transplants and provide a superior
alternative to immunosuppressive agents in the treatment of
autoimmune diseases.
"If those genes are genes that we know a lot about
immunologically, we may be able to understand how it
[tolerance] works," she says.
Even if some of the 1,000 genes turn out to not have a known
role in the immune system, they may still be valuable markers,
because their patterns of expression may be correlated with
different aspects of a disease.
With regard to analysis of gene expression patterns in clinical
specimens, according to Dr. Seyfert-Margolis, the ITN is using
both real time RT-PCR assays and microarray analysis in
parallel on the same clinical specimens.
If successful, it would transform the practice of organ transplants
and provide a superior alternative to immunosuppressive
agents in the treatment of autoimmune diseases
“The clinical investigators see the care that the ITN has taken
in selecting Applied Biosystems. They have a high comfort
level and excitement about sending their specimens to them for
analysis.” says Dr. Seyfert-Margolis.
Genetic markers for immune tolerance
The first step of the project was to hunt for genetic markers for
immune tolerance. The ITN and Applied Biosystems'
scientists relied extensively on the Celera Discovery System™
(CDS) Online Platform. Starting with a list of a few hundred
genes from the ITN, the scientists performed homology
searches of CDS content to generate a comprehensive list of
possible immune tolerance genes by selecting members within
gene families. CDS consolidates high-quality biological content
with visualisation and analysis tools on one web site to make the
job easier. The CDS PANTHER™ Protein Classification
System, for example, categorised the genes’ protein products by
biological process and molecular function. The original list of
genes expanded to 1,000 related genes thought likely to be
involved in the modulation of the immune response.
Finding genetic markers for tolerance
Finding genetic markers for immune tolerance will help
researchers expand the range of therapeutic options available
to clinicians for treating a host of autoimmune diseases,
and different kinds of allergies, as well as for helping to prevent
rejection of transplanted organs. However, the quest to find
such helpful markers has just begun.
"There are not yet any genetic markers for immune tolerance.
If we could find markers that were in fact indicative of
clinical tolerance, it might lead us to a clearer quantitative
definition of tolerance," says Dr. Seyfert-Margolis.
According to Dr. Seyfert-Margolis, a good genetic marker is
one that makes it possible to determine if someone is clinically
tolerant of a foreign antigen. Tolerance may be indicated by a
shift in the gene expression profiles of cytokines, or other genes
may be involved.
"We hope to find [gene expression] patterns that correlate
with clinical changes," says Dr. Seyfert-Margolis. "If we're just
asking if there is going to be an increase in particular cytokines,
those questions we can answer very soon. Whether or not
that means you've achieved tolerance or not I don't know.
All of our studies are two or three years in duration."
One of the biggest challenges for the ITN, but also perhaps one
of the most alluring possible outcomes of these gene expression
studies will be an interpretation of the collected data that leads
to a better understanding of how immune tolerance works.
"Finding the mechanism of action will be difficult.
It will require sharing of information and then having
investigators go back to the bench," notes Dr. Seyfert-Margolis.
Using genetic markers for tolerance
The goals of the ITN, however, go beyond understanding
how tolerance works. They extend to modifying the ways in
which the immune system responds to an antigen. Already,
the ITN is conducting studies of the effects of immune
tolerance in organ transplants, allergies, and autoimmune
diseases such as type 1 diabetes and multiple sclerosis (MS).
For example, for organ transplants, the ITN plans to use
real-time RT-PCR assays to look at a number of genes that it
believes are involved in the prediction of acute rejection events.
"There are a couple of clinical trials coming up in which
clinical investigators will be performing organ transplants in
people who will be given an experimental therapeutic and a
single immunosuppressive agent," notes Dr. Seyfert-Margolis.
"The clinicians will withdraw the immunosuppressive agent
and see if the patients are tolerant. And, in that context,
it's going to be very important to know any piece of
information that would be predictive of an acute rejection
event, before we see pathology."
Any discoveries about immune tolerance in the transplant
studies could perhaps also benefit people afflicted with
autoimmune diseases such as MS, and type 1 diabetes.
a good genetic marker is one that makes it possible to determine
if someone is clinically tolerant of a foreign antigen
Right.
Dr. Vicki Seyfert-Margolis
ITN Executive Director
of Tolerance Assays
Although fairly common, autoimmune diseases are difficult to
define precisely.
"With autoimmunity, you don't know where you are
at the stage of the disease and when people are presenting,"
Dr. Seyfert-Margolis notes. "Autoimmune diseases are
multifactorial and multiple genes are involved. So, these types
of diseases are going to require multiple approaches, multiple
assays, and probably a multivariant sort of analysis."
"I think that the genetics in all of these autoimmune diseases,
such as large studies to look for SNPs (single nucleotide
polymorphisms), are going to be very difficult," she adds.
"Finding molecular markers may help to clarify the situation,
because, to date, there just aren't any good immunological
markers for most of these diseases."
“Autoimmune diseases are multifactorial and multiple genes are involved.
So, these types of diseases are going to require multiple approaches,
multiple assays, and probably a multivariant sort of analysis”
Integrating products and services
Over 22,500 pre-designed, validated, off-the-shelf gene
expression assays are currently available online, with plans
to provide assays for every gene in the human genome
(www.allgenes.com). Approximately two-thirds of the
ITN assays are available as TaqMan Gene Expression Assays
through the Applied Biosystems e-commerce web site
(http://myscience.appliedbiosystems.com). The Custom
TaqMan Assays from Applied Biosystems provided the balance
with custom-made gene expression assays.
For sample preparation, the ITN chose labs that use the
new Applied Biosystems Tempus™ Blood RNA Purification
Tubes for mRNA purification and the ABI PRISM™ 6100
Nucleic Acid PrepStation for sample preparation. The sampleprep labs send the samples to a service laboratory, which in
turn performs quantitative gene expression assays using the
ABI PRISM® 7900HT Sequence Detection System.
The ITN/Applied Biosystems team opted to perform the assays
using the TaqMan Low Density Array format rather than in
tube format. Each of the 384 bubble-wells of the card contains
the primer and probe sets for the gene expression assays.
The service laboratory just adds samples and TaqMan®
Universal PCR Master Mix to the eight channels of the card,
performs a brief centrifugation step, seals the array and
analyses it on the 7900HT real-time PCR system.
Improving therapeutic options
The ITN intends to use the gene expression data derived from
these studies to assess relative expression levels of genes
between normal controls and patients, and in individual
patients at various time points throughout the clinical trials.
The data are expected to track parameters such as disease onset,
disease progression, and patient responses to therapeutics.
Ultimately, this marriage of clinical and genetic sciences
applied to immune tolerance is designed to improve
therapeutic options in organ transplantation, autoimmune
diseases, allergy and asthma.
For further information go to:
http://europe.appliedbiosystems.com/biobeat/itn
INFO
Applied Biosystems Technology:
Informatics
» Celera Discovery System™ Online Platform
» PANTHER™ Protein Classification System
» SQL*LIMS® Software
For more information on:
ABI PRISM® 7900HT
Sequence Detection
System enter: 101
Real-Time PCR
» TaqMan® Gene Expression Assays
» Custom TaqMan® Assays
» ABI PRISM® 7900HT Sequence Detection System
» TaqMan® Low Density Array
Sample Preparation
» ABI PRISM™ 6100 Nucleic Acid PrepStation
» Tempus™ Blood RNA Purification Tubes
ABI PRISM™ 6100
Nucleic Acid PrepStation
enter: 102
Tempus™ Blood RNA
Purification Tubes
enter: 103
Contact
us
Whether you have a question, query or comment on this
article, or any of the others in this issue, please get in
touch with us at: [email protected]
Related articles can
be found on:
Pages 38 & 42
applications
Successful
Connections
Efficient primary structure elucidation of disulfide-bridged peptides using
the PROCISE® cLC sequencing system and the QSTAR® Pulsar system
Stefan Schürch1, Johann Schaller1, Urs Kämpfer1, Lucia Kuhn-Nentwig1, Stefan König2
1. University of Bern, Bern, Switzerland 2. Applied Biosystems, Rotkreuz, Switzerland
INFO
Efficient primary
structure elucidation
of disulfide-bridged
peptides enter: 104
C
omplete de novo primary structure elucidation
of highly disulfide-bridged neurotoxic peptides
from the spider Cupiennius salei is demonstrated,
using a combination of Edman sequencing on the PROCISE
cLC 492 sequencing system and tandem mass spectrometry
on the QSTAR Pulsar hybrid quadrupole time-of-flight
mass spectrometer. The use of these complementary
technologies provides unambiguous analytical data
for successful characterisation of peptides and proteins,
while simultaneously extending the range of applications.
Introduction
N-terminal amino acid sequence analysis by Edman
degradation has been the working-horse for the de novo
sequence determination of peptides and proteins for many
years. Modern sequencing systems based on the Edman
chemistry provide high sensitivity and enable fully automated
analyses. Due to the unambiguous sequence data generated,
Edman sequencing is still the reference technology for
complete de novo protein sequencing. As a further key
technology, mass spectrometry has entered the field of
bioanalysis within the last decade, enabling rapid protein
identification. The impressive resolving power and mass
accuracy of modern mass spectrometric instrumentation
offers great potential for elucidation of structural details.
The combination of Edman sequencing and tandem mass
spectrometry is an efficient approach to the primary structure
elucidation of complex, highly disulfide-bridged spider toxins.
Methods
Edman sequencing: The sequences of two reduced and
alkylated neurotoxic peptides CSTX-1 (8,351.90 Da) and
CSTX-9 (7,530.25 Da) were determined by Edman
degradation on a Procise cLC 492 protein sequencer1,2,3.
The overlaps were secured by a combination of enzymatic
cleavages with Asp-N, chymotrypsin, and immobilised
trypsin (figure 1). The disulfide-containing peptides were
generated by cleaving native CXTX-1 and CSTX-9 with
immobilised trypsin.
Electrospray tandem mass spectrometry: Identification of the
disulfide-bridge pattern of the peptides was performed by
nano-electrospray tandem mass spectrometry on the QSTAR
Pulsar hybrid quadrupole time-of-flight mass spectrometer 2,4.
The purified tryptic peptides, consisting of four to five chains
cross-linked by the original disulfide bridges, were subjected to
MS and MS/MS analysis.
Results and Discussion
The complete sequences of CSTX-1 and CSTX-9 with the
corresponding overlaps are shown in figure 1. The proximity of
the cysteine residues and the absence of suitable cleavage sites
within the disulfide-containing sequences caused the classical
approaches for elucidation of the disulfide-bridge pattern to fail.
Consequently, the basically unspecific gas-phase dissociation of
the disulfide-linked peptides by tandem mass spectrometry is
an attractive alternative to the classical procedures.
Identification of the disulfide bridge pattern was based on
observation of characteristic fragment ions generated by
dissociation of the peptide bonds adjacent to the cysteines, as
illustrated for CSTX-9 in figure 2. The identification of the
disulfide-bridge pattern of CSTX peptides by tandem mass
spectrometry represents a valuable alternative to traditional
methods for elucidation of complex molecular structures.
Modern high-performance instrumentation, such as the
QSTAR Pulsar mass spectrometer, greatly enhance the
potential of this technique, since a wide range of information
is obtained from a single experiment using minute amounts of
sample only. Sample preparation is essentially reduced to a
purification step, thus, avoiding the risk of potential structural
alteration, e.g. disulfide scrambling.
References:
1. L. Kuhn-Nentwig, J. Schaller, W. Nentwig, Toxicon, 1994, 32, 287-302.
2. J. Schaller, U. Kämpfer, S. Schürch, L. Kuhn-Nentwig, S. Haeberli,
W. Nentwig, Cell Mol. Life Sci., 2001, 58, 1538-1545.
3. J. Schaller, L. Kuhn-Nentwig, S. Schürch, U. Kämpfer, J. Müller,
W. Nentwig, Chimia 2001, 55, 1058-1062.
4. S. Schürch, J. Schaller, U. Kämpfer, L. Kuhn-Nentwig, W. Nentwig,
Chimia 2001, 55, 1063-1066.
Figure 1.
Amino acid sequences of CSTX-1 (top) and CSTX-9 (bottom) determined by Edman
degradation. Positions sequenced are indicated by horizontal bars. Up to 53 amino acids
were sequenced in a single run.
Conclusions
The results demonstrate that Edman sequencing and
tandem mass spectrometry are by no means competing
techniques. These two techniques can be considered as
complementary tools, each one offering its particular
advantages. Edman sequencing is a straightforward and
efficient approach to the determination of amino acid
sequences of peptides and proteins, whereas tandem mass
spectrometry exhibits its ultimate potential for the
elucidation of structural details. If both techniques are
available to the researcher, bioanalytical problems are
solved most accurately and efficiently.
Figure 2.
Product ion spectra are characterised by abundant y-type ions due to cleavage of the
unbridged portions of the peptide chains. Bridge-defining fragment ions of higher charge
state are identified unambiguously due to the high resolving power and mass accuracy
of the QSTAR® Pulsar mass spectrometer. The disulfide-bridge pattern corresponds to
the inhibitor cystine knot (ICK) structural motif.
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applications
Determination
of the
Doping Agent
Tetrahydrogestrinone
by LC-APCI-MS/MS
Abstract
he designer steroid tetrahydrogestrinone (THG) was identified in urine samples of elite athletes in the
doping control laboratory in the United States of America. Hence, all doping control samples have to be analysed
in this regard requiring reliable and robust assays, which have been established employing liquid
chromatography/tandem mass spectrometry. Utilising an API 2000™ LC/MS/MS System, THG was determined at
concentrations of 2 ng per mL of human urine by means of multiple reaction monitoring of characteristic product ions.
T
Introduction
Gestrinone (figure 1, 1) is a drug administered in cases
of endometriosis1. Its hydrogenation at the ethinyl residue
at carbon 17 results in a steroid hormone termed
tetrahydrogestrinone (THG), the physiological effects of
which have not been investigated until now. The identification
of the designer steroid THG (figure 1, 2) by researchers of
the doping control laboratory of the University of California,
Los Angeles (UCLA), has triggered an avalanche in sport as
well as the scientific world2-3. The hydrogenation of gestrinone
gives rise to a compound related to anabolic steroids, and the
shift of four amu compared to gestrinone invested it with
invisibility for conventional multiple reaction monitoring
(MRM) screening procedures. With the knowledge of structure
and molecular weight of THG and its synthesis as reference
compound, an identification in human urine has become
feasible by LC-APCI-MS/MS, and its determination is
included in established procedures covering anabolic steroids
such as trenbolone and structural analogues.
Doping control urine samples are tested for the presence of THG
and other prohibited compounds in a screening procedure
employing two principal sample preparation steps
INFO
API 2000 LC/MS/MS
System enter: 105
Related article can
be found on:
Page 16
Acknowledgements
Mario Thevis and
Wilhelm Schänzer,
Institute of
Biochemistry, German
Sport University,
Cologne, Germany
Methods
Doping control urine samples are tested for the presence of
THG and other prohibited compounds in a screening
procedure employing two principal sample preparation steps.
Urine specimens are treated with β-glucuronidase from E. coli
in order to hydrolyse glucuronic acid conjugates of THG and its
metabolites, and analytes of interest are extracted from urine
with n-pentane. The organic layer is separated, evaporated to
dryness, the residue is reconstituted in methanol, and the
sample is analysed by LC-APCI-MS/MS. The analytical
instrumentation consists of an Agilent (Waldbronn, Germany)
1100 Series HPLC equipped with a Merck (Darmstadt,
Germany) Purospher Star 18e column (4 x 55 mm, particle size
3.5 µm), which is interfaced to an API 2000 triple quadrupole
mass spectrometer. The API 2000 system is operated in the
positive MRM mode, and for THG two diagnostic ion
transitions (m/z 313 – 241, m/z 313 – 199) are recorded.
Figure 1.
Chemical structures
of gestrinone
(1, mol wt = 308),
and tetrahydrogestrinone
(2, mol wt = 312).
Figure 2.
ESI-product ion spectrum
of (M+H)+ of gestrinone
(m/z 309, a) and
tetrahydrogestrinone
(m/z 313, b) at collision
offset voltages of 40.
Results and discussion
THG was synthesised from gestrinone using an adequate
catalyst and hydrogen. The reference compound was used
to optimise instrument parameters such as declustering
potential, collision energies and exit potentials of the
API 2000 system, resulting in sufficiently sensitive screening
and confirmation procedures.
In figure 2, the product ion mass spectra of gestrinone and
THG are shown, both containing the characteristic fragment
ions at m/z 241 and 199 after ESI and collisionally activated
dissociation. These ions are targets of MRM analyses of doping
control urine samples, and, in combination with retention
times, signals reliably indicate the presence or absence of THG.
A confirmatory analysis in case of suspicious results is based on
at least three diagnostic ion transitions that have to meet the
criteria of the International Olympic Committee (IOC) and
World Anti-Doping Agency (WADA). Figure 3a demonstrates
a typical extracted ion chromatogram of a urine sample fortified
with 2 ng of THG per mL of urine, which, in comparison
to a blank specimen in figure 3b, contains distinct peaks at
13.5 min at the respective ion transitions.
Since the discovery of THG in October 2003, numerous
samples that have been analysed and reported already,
were re-analysed in terms of the presence of THG, and the
assay based on LC-APCI-MS/MS has proven to be a robust
tool generating reproducible results for the detection and
identification of this particular designer steroid in combination
with other prohibited drugs.
Figure 3.
Extracted ion
chromatograms of a)
urine sample fortified with
2 ng/mL of THG, and b)
blank urine specimen.
References
1. Martindale – The Extrapharmacopoeia, 30th edition,
James E. F. Reynolds (Ed.), Pharmaceutical Press, London, 1993, p. 1185
2. Knight, J. Nature 425, 752 (23 Oct 2003) News
3. Knight, J. Nature 426, 114 - 115 (13 Nov 2003) News Feature
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Characterisation of the proteome of synaptic
proteins from rat brain using cICAT® ReagentLC MS/MS in combination with 2D-gel
electrophoresis MS and MS/MS
1
2
Ka Wan Li and Martin P. Hornshaw
1. Faculty of Earth and Life Science, Free University, Amsterdam, The Netherlands. 2. Applied Biosystems, Warrington, UK
INFO
Cleavable ICAT
enter: 106
Related article can
be found on:
Page 34
Overview
he brain is the most complex and dynamic of all
organs with a high degree of computation capability
that enables an animal to perceive and integrate
information and to respond to environmental and physiological
inputs. Central to the neuronal circuitry of the brain is the
extensive synaptic connections via which neurotransmission
occurs. Synaptic neurotransmission involves the release of
transmitter from the pre-synaptic compartment, and the
activation of transmitter receptors and signal transduction
cascades in the post-synaptic density (PSD) of the synaptic
spine. It is generally accepted that information storage in the
brain is established via structural and molecular changes of
synapses, which arise at least in part from the alteration
of protein constituents in the synapse. As a first step towards
the understanding of synaptic function and plasticity,
we applied 2-D gel-based and cleavable Isotope Coded Affinity
Tag (cICAT)-based technologies to characterise the proteome of
this organelle1.
T
Approach and Results
Purification of the PSD
We used a two-step detergent extraction in conjunction with a
sucrose gradient ultra-centrifugation to purify the PSD fraction,
which also contains a low level of some pre-synaptic proteins.
Forebrains from adult rats were homogenised in a sucrose
solution and centrifuged at low speed to remove the nucleus
and the intact cells. The supernatant was centrifuged at a higher
speed to obtain a P2 pellet. Synaptosomes, which contain
the pre- and post-synaptic compartments, were isolated from
this P2 pellet by ultra high-speed centrifugation in a sucrose
gradient, and then extracted twice with Triton-X 100.
After a final high-speed centrifugation the PSD fraction,
which constitutes a compact detergent insoluble protein
complex, was recovered from the pellet phase.
Separation/fractionation of the PSD
and analysis by mass spectrometry
To identify the proteins of the PSD fraction, two separation
approaches have been followed, namely 2-D gel electrophoresis
and cICAT Reagent liquid chromatography, which were
followed by mass spectrometric analyses.
The identification of the proteins is based on the MASCOT score
of the peptide mass fingerprint and of the daughter
ion spectra of the tryptic peptides
2-D gel electrophoresis and sample
preparation for mass spectrometry
For 2-D gel electrophoresis separation of proteins, samples
were solubilised in lysis buffer for 30 min and then used for the
rehydration and simultaneous loading of the proteins to the
IPG strip, which was then focused for 65000V/hr. The second
dimension separation was run overnight in 11% gels.
After electrophoresis, gels were fixed and stained using colloidal
Coomassie brilliant blue G-250. All the visible protein spots
from the Coomassie blue stained gel were manually excised,
destained and dried. The gel pieces were then rehydrated in 8 µl
trypsin solution (20 µg/ml). After incubation overnight at room
temperature, 0.5 µl of the incubation buffer was pipetted to the
MALDI plate and mixed with 1 µl α-cyano-4-hydroxycinnamic
acid. The samples were analysed with a MALDI TOF/TOF
mass spectrometer, the 4700 Proteomics Analyzer with
TOF/TOF™ optics from Applied Biosystems. In cases where the
mass spectrometric signals were weak, the remaining incubation
buffer was loaded into a C18 Ziptip® (Millipore) according to the
protocol provided. The bound peptides were eluted from
the Ziptip using 1.0 µl of α-cyano-4-hydroxycinnamic acid,
which was directly deposited onto the MALDI plate.
The trypsin auto-digestion fragments at the protonated masses
of 842.510 Da and 221.104 Da, were used as internal
standards for the calibration of the MS spectra. All the mass
spectra were searched against the NCBI database, using online
MASCOT® software.
Mass spectrometry identification results
by 2-D gel electrophoresis
The identification of the proteins is based on the MASCOT
score of the peptide mass fingerprint and of the daughter ion
spectra of the tryptic peptides. In total 250 protein spots were
analysed. Interestingly, in about 35% of the cases single proteins
are present in 2 or more spots. As an example synapsin II is
detected in 14 spots, from which two groups can be clustered
together. The group of proteins that are smaller and generally
more acidic represents one of the alternative spliced forms of
synapsin II, named synapsin IIb (figure 1). Within both groups,
trains of spots that differ in pH can be discerned. This can be
explained in part by the differential phosphorylation of
multiple phosphorylation sites, through which a progressive
decrease in isoelectric point of the protein resulted. In this
respect 14 serine, 4 threonine and 2 tyrosine sites on synapsin
IIb are predicted to be the phosphorylation sites.
Although functional implications of the dynamic and
differential regulation of the phosphorylation of synapsins have
been well documented, due to the complexity of the events
many of the phosphorylation sites and their combinatorial
existence on single protein remain to be characterised.
Other posttranslational modifications of PSD proteins have also
been reported and may contribute to the appearance of some of
the multiple spots on the 2-D gel. Table 1 summarises the
list of proteins that show multiple spots (>5 spots per protein)
on the 2-D gel.
Left.
Members of the
neuroproteomics group
at the faculty of Earth
and Life Sciences of the
Vrije Universiteit
Amsterdam.
From left to right:
K.W. Li, C.R. Jimenez,
A.B. Smit, Y. Gouwenberg,
R.C. Van Der Schors,
Z. El Filai.
cICAT Reagent derivatisation, separation and
mass spectrometry identification results
by cICAT Reagent/liquid chromatography
2-D gel electrophoresis–based methodology is a widely used
technique for proteomics studies. However, in our study some
major PSD proteins are under-represented, for example,
glutamate receptor subunits are not detected although they are
known to be highly enriched in the PSD fraction. To address
this problem, we performed an alternative experiment that
combined the cICAT Reagent derivatisation/purification
protocol of the tryptically digested PSD to nano-liquid
chromatography-electrospray tandem mass spectrometry.
Briefly, the purified sample was labelled with the cICAT
Reagent and then digested with trypsin to produce tryptic
peptides. The cICAT Reagent generates cysteine-containing
peptides that can be affinity selected from non-derivatised
peptides using avidin chromatography, substantially reducing
sample complexity. After elution from the avidin affinity
column, the cysteine-containing peptides were resolved by a C18
column (75 µm id, flow rate 200 nl/min, LC Packings) and
sprayed on-line into an electrospray tandem mass spectrometer
from Applied Biosystems (QSTAR® Pulsar i LC/MS/MS
system) that automatically performs information dependent
tandem MS acquisitions. The spectra were searched against the
Pro ICAT engine. In addition to the cytosolic proteins and
cytoskeletal proteins, the cICAT Reagent LC-MS/MS
experiment allows the detection of hydrophobic proteins
such as the ionotropic glutamate receptors and ion channels,
large proteins (plectin > 500 kDa) and basic proteins
(ribosomal protein > pH 10) that were missed by the 2-D gel
approach. On the other hand, the different forms of
posttranslationally modified proteins, as trains of protein
spots on the 2-D gel, cannot be detected.
Concluding remarks
2-D gel and cICAT Reagent LC based proteomics approaches
are complementary and detect overlapping sets of proteins
(figure 2). Together, we show that the PSD contains a high
diversity of functional classes of protein. This is in agreement
with previous studies that showed the possible extension of the
PSD into the synaptic spine compartment; the spine apparatus,
polyribosomes and specialised endocytic zones are found
located in close vicinity to the PSD that together with other
“cytosolic” proteins such as glycolytic enzymes and signalling
enzymes may be interconnected to the PSD by actin filament.
Together, we postulate that the PSD is a complex organelle
harbouring diverse physiological functions, which puts the
PSD into a central position for the autonomous functioning of
the synaptic spine.
References
1. Ka wan Li, Martin P. Hornshaw, Roel C. Van der Schors,
Rod Watson, Connie, R. Jimenez, Yvonne Gouwenberg,
Eckart D. Gundelfinger, Karl-Heinz Smalla, Stephen Tate,
August B. Smit (2004) Proteomic analysis of rat brain
postsynaptic density: implications of the diverse protein
functional groups observed for the integration of synaptic physiology.
J. Biol. Chem. 279, 987-1002.
Figure 1.
Synapsin IIb (indicated
by arrows) appeared
as multiple spots on
the 2-D gel.
Figure 2.
Functional grouping of
PSD proteins identified
by the 2-D gel based (A)
and cICAT Reagent LC
based (B) approaches.
Protein name
Number of spots
SAP90/PSD-95
6
Vesl-1L/Homer 1
6
CaM kinase II α
6
Actin β
10
Tubulin α
13
Tubulin β
18
Neurofilament, light
10
Internexin α
11
Glyceraldehyde-3-phosphate dehydrogenase
7
Aldolase
6
Synapsin I
9
Synapsin II
14
Table 1.
Proteins from the PSD
fraction that are detected
on the 2-D gel as
multiple spots.
About 35% of the distinct
proteins were represented
by two or more protein
spots. Here, only those
that are presented as
6 or more spots on the
2-D gel are shown.
applications
Peptidomics
®
Differential Analysis and Inventory
of Human Cerebrospinal Fluid
Acknowledgement
Dr Peter SchulzKnappe, BioVisioN,
Germany
Peptide Identification
For identification purposes sample amounts of 4-20 ml
CSF were prepared according to the DPD analysis,
which was possible due to our scalable preparative process.
Peptide identification was performed by means of
offline nanoESI-MS/MS (QSTAR® Pulsar System from
Applied Biosystems/MDS SCIEX, Concord, Canada) or
online nanoHPLCESI-MS/MS. Generated MS/MS spectra
were mass deconvoluted, and submitted to the MASCOT®
database engine.
Peptides and small proteins, such as hormones,
cytokines and growth factors play an active
role in many physiological processes
elution time (fractions 1-96)
Related article can
be found on:
Page 24
Relative molecular mass/charge (750 - 15,000)
Material and Methods
Differential Peptide Display® (DPD)-Analysis Peptides were
extracted from raw samples of CSF (0.5ml each) and
fractionated in 96 fractions by reversed-phase chromatography.
All fractions were measured by Matrix-Assisted Laser
Desorption/Ionization Time-Of-Flight mass spectrometry
(MALDI-TOF-MS, Voyager-DE™ STR, Applied Biosystems,
Framingham, MA, USA) resulting in a peptide mass
fingerprint with each peptide precisely characterised by its
relative molecular mass, elution/retention time during
chromatography and signal intensity. The 96 MALDI-MS
spectra from each sample were assembled and visualised
as 3-dimensional diagrams similar to “2D-Gel Pictures”
(figure 1). This was done by Spectromania®, a purpose-built
software package for data visualisation and sophisticated
statistical analysis of multiparametric data sets1.
Relative molecular mass/charge (1,200 - 3,000)
elution time
For more information
visit:
http://www.biovisiondiscovery.de/set_all.htm
P
Figure 1.
Peptide master maps representing the CSF peptidome from healthy
controls (A) and patients with primary CNS lymphoma (PCNSL; B).
The comparison of the master maps resulted in a heat map.
Shown is a detail with the relative mass range between 1,200 and
3,000. The average intensities from both sets were compared for every
single signal position calculating the absolute difference. Signals with
the same intensity in both sets are represented by white spots, whereas
red and blue colour indicate stronger signals from PCNSL patients and
controls, respectively2.
Relative molecular mass/charge (750 - 15,000)
elution time (fractions 1-96)
INFO
Introduction
eptides and small proteins, such as hormones,
cytokines and growth factors play an active role in
many physiological processes. Moreover, as products
of protein degradation and processing, peptides are part of
proteometabolism. Thus, they can reflect disease-related
changes in an organism’s homeostasis in several ways. A peptide
source of high diagnostic value is human cerebrospinal fluid
(CSF), in which peptides are mainly derived from the central
nervous system (CNS) or from blood.
Results
DPD-guided identification of peptides in human CSF was
carried out to investigate several diseases of the CNS. For example,
this approach led to novel biomarker panels in the field
of Alzheimer’s Disease (AD3) and oncology (PCNSL2):
Among others, the peptide-biomarker panel of AD consist
of fragments derived from neurosecretory protein VGF,
osteopontin and complement C33. As a major difference,
a fragment of serum albumin precursor (ALBU 25-48) (figure 1)
was identified in the PCNSL-study. The increase of the
albumin fragment resulted from a bloodbrain barrier disruption,
typically occurring in these tumour patients.
Peptide Inventory of human CSF
The separation into 96 fractions revealed the presence of more
than 6,000 signals in the mass spectrometric analysis, which are in
part redundant due to multiple charge states, oxidation products
or analytical derivatives like sodium adducts, loss of water etc.
Here, the Peptide Inventory was created by analysing every second
fraction using nanoHPLC-MS/MS or offline nanoESI-MS/MS.
Table 1 gives an extract of the most abundant protein
precursors with regard to the number of identified fragments.
In addition to their relevance as diagnostic markers, the peptides
represent insights into the proteometabolism in CSF. The observed
cleavage sites with regard to the precursors indicate different
enzymatic processing motifs in CNS, CSF or in blood.
For example, we observed that several fragments were released
from their precursor by cleavage at mono- and dibasic sites.
This is indicative for neuroendocine prohormone processing
and in accordance with results found by other groups4.
Summary and Conclusions
» A comprehensive Peptide Inventory of a biological
source like CSF the determination of prominent landmarks
allows a general understanding of the biological source to
be achieved
» Due to our highly reproducible and scalable process it is possible
to assign Peptide Inventory data to DPD data and vice versa
» DPD technology can be used for the systematic analysis and
visualisation of the peptide content of CSF using small sample
amounts
» Peptidomics technologies allow the identification of
peptides with intrinsic biological relevance important for
diagnosis and/or therapy
Table 1.
No.
Protein precursor
Accession No.
No. of identified
fragments
1.
Neurosecretory protein VGF precursor
015240
35
2.
Serum Albumin precursor
P02768
22
3.
Secretogranin I precursor
P05060
20
4.
Fibrinogen alpha/alpha E-Chain precursor
P02671
17
5.
Transthyretin precursor
P02766
15
6.
Osteopontin precursor
P10451
11
Identified peptides were annotated on a CSF master map,
which is shown in figure 2.
Figure 2.
Annotated peptide master map of human CSF.
Each symbol represents an identified peptide.
References
1. P. Schulz-Knappe, H.-D. Zucht, G. Heine, M. Juergens, R. Hess,
M. Schrader, Comb. Chem. High Throughput Screen. 4 (2001) 207.
2. G. Heine, H.-D. Zucht, M.U. Schuhmann, K. Buerger, M. Juergens,
M. Zumkeller, C.G. Schneekloth, H. Hampel, P. Schulz-Knappe, H. Selle,
J. Chromatogr. B 782 (2002) 353.
3. Patent No. WO 02/082075 A2, WO 02/090974 A2, WO 03/048775 A2.
4. M. Stark, O. Danielsson, W.J. Griffiths, H. Jörnvall,
J. Johansson, J. Chromatogr. B 754 (2001) 37.
Contact
us
applications
Determination
of residues of
chloramphenicol
in milk and milk products
using HPLC/MS/MS
Klara Oresnik, Milojka Bedek and Julija Bericnik-Vrbovsek,
ERICo, Ecological Research & Industrial Cooperation, Velenje, Slovenia
Gabor Balizs, Federal Institute for Risk Assessment, Berlin, Germany
André Schreiber, Applied Biosystems, Darmstadt, Germany
Abstract
n the European Union the use of chloramphenicol
(CAP) as a veterinary drug for food producing animals is
prohibited. For that reason a rapid and sensitive method
for the identification and quantification of CAP in milk and
milk products using LC/MS/MS has been developed.
I
Blank milk samples were spiked with CAP in the
concentration range of 0.05 - 1.5µg/L. The clean up is
based on extraction with acetonitrile, followed by defatting
with chloroform and Solid Phase Extraction on a
styrene-divinyl-benzene polymer cartridge. The final extract
was analysed by LC/MS/MS using negative Electrospray
ionisation. The method was validated according to the new
EU regulation (657/2002/EC).
Introduction
Chloramphenicol (CAP), an antibiotic was formerly used
widely in veterinary medicine, due to its broad range of activity
and low cost. The treatment of animals used for production of
food with CAP is prohibited in the European Union and
United States because of the side effects in humans,
in particular the dose independent fatal aplastic anaemia.
a rapid and sensitive method for the identification and
quantification of CAP in milk and milk products
using LC/MS/MS has been developed
Thus, it is necessary to control residues of CAP in edible
tissues, like milk and milk products.
Methods to detect CAP in biological matrices especially in
milk described in the literature include immunoassays1,
GC/MS2-4 and LC5-6. The immunological methods are
suitable for screening purposes, whereas mass spectrometric
methods are used for confirmation analysis. LC/MS can
be used without derivatisation, while silylation is necessary
for GC/MS.
Therefore, a rapid and sensitive method for the identification
and quantification of CAP in milk and milk products
using LC/MS/MS has been developed. The advantage of
the described method is its high recovery and the short time
of analysis.
Experimental
The sample preparation procedure (based on reference 7) is
presented in figure 1. (more details of clean up will be
published soon). HPLC separation was carried out using an
Agilent 1100 HPLC system with degasser, binary pump and
autosampler, a Phenomenex column LUNA 50x2mm (3µm)
with Security Guard column, and a gradient of water + 2mM
ammonium acetate (eluent A) and methanol (eluent B). A flow
rate of 350µL and gradient profile (A/B) of 0 min 55/45,
5 min 10/90, 10 min 10/90, followed by re-equilibration was
used. A sample volume of 50 µL was injected.
Detection was performed on an API 3000™ LC/MS/MS
system with TurboIonSpray® Source in negative ion mode and
Multiple Reaction Monitoring (MRM). The following MRM
transitions (Collision Energy) were used for quantitation:
321/152 (-24V), 321/257 (-12V), 321/194 (-16V),
and 321/176 (-20V). Under these conditions, MRM 321/152
was the most intense transition and, thus, used as quantifier
while other transitions were used as qualifiers (figure 2).
Chloramphenicol
This method is able to identify and quantify residues
of chloramphenicol in milk and milk products
in lower ppb concentration range
Figure 1.
Sample clean up for milk
and milk powder samples
The validation was done according to the criteria of the
Commission Decision 657/2002/EC8.
The deviation of retention times between standard and samples
were lower than 2.5% (the average retention time of standard
2.54 min and 2.56 min of the milk samples).
A system of identification points must be used according
to 657/2002/EC when mass spectrometric detection
follows chromatographic separation. For the confirmation
of substances listed in Group A of Annex I according
to 96/23/EC9, like chloramphenicol, a minimum of
4 identification points is required. According to this definition,
4 identification points earned using LC/MS/MS with
1 precursor and 2 product ions. The presented method detects
4 product ions, thus, performance criteria for confirmation are
fulfilled. Relative ion intensities are used to qualify results of
quantitation. The deviation of relative ion intensity in spiked
milk samples (vs. standard) was lower than 10%.
Figure 2.
Chromatogram
containing 4 MRMs
of Chloramphenicol
(1µg/mL in milk)
The calculated limit of decision (cca) was found at 0.03 µg/L,
the detection capability (ccß) at 0.1 µg/L, and the recovery
at 70% with a coefficient of variation of 5%. Coefficient of
correlation of > 0.99 for all 4 transitions were determined
(7 points of calibration and 10 replicates).
Conclusion
This method is able to identify and quantify residues of
chloramphenicol in milk and milk products in lower
ppb concentration range. Due to its high specificity
the LC/MS/MS assay fulfils criteria of the EU regulation
657/2002/EC.
References
1. D. Arnold and A. Somogyi: J. AOAC 68 (1985) 984
INFO
2. G. Balizs and D. Arnold: Chromatographia, 27 (1989) 489
The API 3000 LC/MS/MS System enter: 107
3. P.Fürst, C.Krüger, H.A.Meemken, and W.Groebel: D. Lebensmittel
Rundschau 84 (1988) 108
4. P.J.Kijak: J. AOAC 77 (1994) 34
To download related application notes, visit:
http://www.appliedbiosystems.eu.com/mk/get/ABSMPDFREG
5. A.Pfenning, S.Turnipseed, J.Roybal, M.Madson, R.Lee and J.Storey:
U.S. Food and Drug Administration, Denver, LIB No.4294, 19 (2003)
6. A.Gantverg, I.Shishani and M. Hoffman: Analytica Chimica
Acta 483 (2003) 125
7. V. Hormazabal, M. Yndestad: J. Liq. Chrom. & Rel. Technol.
24 (2001) 2477
8. Community Decision 657/2002/EC
Related article can be found on: Page 10
Contact
us
Whether you have a question, query or comment on this article, or any of the others
in this issue, please get in touch with us at: [email protected]
9. Council Directive 96/23/EC of 29 April 1996
applications
Applied Biosystems
technology
arms researchers
in the battle against
SARS
by Mark Springer
BioBeat®
Online Magazine
www.biobeat.com
I
n February 2003, a mysterious illness resembling
pneumonia began to spread rapidly through different
parts of Asia. At first, it was thought that an outbreak of
bird flu virus, similar to the one that killed six people in 1997,
was to blame1. However, the next month, the world was
introduced to the name Severe Acute Respiratory Syndrome,
or SARS and the mysterious disease quickly spread from
Asia to six continents in a matter of weeks.
In fact, just a few months after a name was given to the
mystery disease, researchers armed with products from
Applied Biosystems and technologies had already developed
the first SARS diagnostic test, and identified key viral proteins
that represent potential drug targets. Identification of the virus
responsible for SARS, and high-throughput DNA sequencing
of the viral genome have helped researchers to better
understand the origins of the disease
SARS spread quickly
In late 2002 sporadic cases of a mysterious respiratory
illness first began to occur in Guangdong Province, China.
About three months later the disease had spread to several
different countries through international air travel.
At that time, about a dozen people – all of whom were
believed to have contracted SARS from a Guangdong
Province doctor while they all stayed on the same floor of
the Metropole Hotel in Hong Kong – left Hong Kong and
fanned out to different parts of the world2. It then spread to
Singapore, Canada, and Vietnam. And it was in Vietnam
where the illness claimed the life of Dr. Carlo Urbani, a World
Health Organization (WHO) physician, who was the first one
to identify the outbreak while treating one of the first patients
stricken with SARS, a sick traveller from Hong Kong2.
The world was introduced to the name Severe Acute Respiratory Syndrome,
or SARS and the mysterious disease quickly spread from
Asia to six continents in a matter of weeks
Above.
Dr. Christian Drosten,
Bernhard Nocht Institute
for Tropical Medicine,
Hamburg, Germany
Public health efforts reduce risk,
but danger remains
Today, thanks to global public health efforts and good
infection control practices around the world, new SARS
cases have disappeared, with only a few infections
being reported since July 20033. However, the apparent
disappearance of SARS does not necessarily mean the
disease will not strike again3.
Weekend discovery
In March 2003, Dr. Christian Drosten, Bernhard Nocht
Institute for Tropical Medicine, Hamburg, Germany worked
non-stop for an entire weekend to determine the genetic
makeup of the virus responsible for SARS from a cell culture
infected with material from a SARS patient sample.
Based on the viral genetic sequences he discovered,
he developed a real-time RT-PCR (reverse transcriptionpolymerase chain reaction) assay that was subsequently
incorporated into the first SARS diagnostic test, a test that is
currently being marketed by Abbott labs and distributed
through its molecular diagnostics alliance with Celera
Diagnostics, a joint venture of Applied Biosystems and Celera
Genomics, two business groups of the Applera Corporation4.
Developed into a commercial kit by the German company
Artus GmbH, and based on the technique of real-time PCR,
the SARS diagnostic test detects the presence of the SARS viral
genetic material in patient blood and tissue samples.
The diagnostic test
The SARS diagnostic test is based on a real-time RT-PCR
assay that uses probes and primers made from DNA
fragments selected from regions of the SARS-associated
coronavirus (SARS-CoV) genome. Researchers at the Bernhard
Nocht Institute used an ABI PRISM® 3100 Genetic Analyzer
and BigDye® Terminator sequencing reagents for all
DNA sequencing reactions needed for development
of primers and probes used in the real-time RT-PCR assay.
The ABI PRISM® 7000 Sequence Detection System, also from
Applied Biosystems, automated many of the real-time RT-PCR
assays used in the development of the diagnostic test5.
Test can confirm, but not rule out
"The only way to use the PCR diagnostic test is to confirm
suspected [SARS] cases, not to rule out the disease," explains
Dr. Drosten. "If you find a positive signal, then you can say
that it's really SARS," says Dr. Drosten. "WHO asks you to use
a second test for confirmation, but that's usual laboratory
procedure that you would also do with many other diseases to
confirm a positive PCR result with a second PCR test."
“The first thing that I did was a cross titration to
look at different combinations of primers.
After that, we optimised the reagents.“
For instance, sputum samples showed much higher
concentrations of viral RNA than did serum samples5.
ABI PRISM 3100
Genetic Analyzer
"The problem is that the clinicians are a little bit afraid of
taking these samples of deep respiratory materials from SARS
patients, because when you have a SARS patient cough deep
from his lungs, he [or she] will produce quite a lot of virus in
the air that will produce aerosols and put the clinicians at risk
of becoming infected," says Dr. Drosten.
Positive past experience dictated choice
of Applied Biosystems technology
To detect the presence and amount of virus detected in
different clinical specimens, researchers used fluorescentlylabelled coronavirus-specific probes and primers,
and automated many of the real-time RT-PCR assays
using the 7000 system.
ABI PRISM 7000 Sequence
Detection System
Dr. Drosten is a long-time user of sequence detection systems
from Applied Biosystems and his lab is equipped with both a
7700 system and a 7000 system, instruments that he regularly
uses to automate RT-PCR assays.
"We use the 7000 system for a lot of our routine assays that are
operated by technicians," he continues. "It's very easy to use.
It's quite easy to evaluate the data that you collect."
Sequence detection systems help
to quantify SARS virus
In addition, the SARS diagnostic test also measures different
levels of expression of the viral genetic material in different
kinds of clinical specimens.
"Another reason [that we use Applied Biosystems sequence
detection systems] is that the thermal cycler inside this system
is one of the most renowned cyclers, the GeneAmp® PCR
System 9700. And, in the previous model, the 7700 system,
the GeneAmp® PCR 9600 system, a thermal cycler that has
been in operation for about 15 years. So, there is really reliable
technology that you have as a basis for these machines,"
Dr. Drosten notes.
When the researchers quantified amounts of viral RNA by
performing a real-time RT-PCR assay, they found answers to
many of their questions. Real-time RT-PCR assays revealed
significant disparity in the concentration of SARS-associated
coronavirus in different kinds of samples containing viral
genetic material5.
Virus identified through random
amplification approach
Having only part of the genomic sequence of the virus,
the researchers identified the virus by randomly amplifying,
and then sequencing fragments of the virus genome obtained
from tissue cultures and supernatant.
GeneAmp PCR
System 9700
Page 20
For example, similarities in the structure of the viral main
proteinase (also called 3CLpro), an essential component of the
SARS-CoV replication machinery, to a protein of similar
function in rhinoviruses, viruses that can cause the common
cold, attracted the attention of researchers at the University of
Lübeck, in Germany7.
These researchers found that it might be possible to modify
an available cold virus drug so that it inhibits the functioning
of the SARS-CoV proteinase, an enzyme that plays a key
role in controlling the replication of SARS-CoV. The structure
of the SARS-CoV proteinase resembles the structure of a
cold virus protein previously found to be inhibited by
rhinovirus inhibitors7.
Above.
Coronavirus
An electron microscopic
photo of the Frankfurt,
Germany strain of
the SARS-associated
coronavirus (SARS-CoV).
Photo captured by
Professor Herbert
Schmitz of the
Bernhard Nocht Institute
for Tropical Medicine.
Permission for use
of photo granted by
Dr. Christian Drosten.
INFO
BigDye Terminator
Sequencing Reagents
enter: 108
ABI PRISM 3100 Genetic
Analyzer enter: 109
Sequence Detection
Systems from
Applied Biosystems
enter: 110
Thermal Cyclers from
Applied Biosystems
enter: 111
Related article can
be found on:
Page 38
To design the primers for use in the real-time RT-PCR
assay, Dr. Drosten used Primer Express® Software from
Applied Biosystems. He sought the best possible combination
of forward and reverse primers for the assay.
"The first thing that I did was a cross titration to look at
different combinations of primers. After that, we optimised
the reagents."
Researchers identify mystery virus
as a coronavirus
A team of researchers led by Dr. Malik Peiris, and his
colleague Dr. K.Y. Yuen, both from the Department of
Microbiology at the University of Hong Kong discovered a
cluster of viral particles lurking amidst a group of dying cells.
They recognised the crown-like spherical membranes
surrounding each viral particle as the trademark signature
of a family of viruses known as coronaviruses, perhaps best
known for being one of the virus families responsible for
the common cold2.
The coronaviruses are RNA viruses which are less stable than
DNA viruses and tend to mutate more frequently6.
Simultaneously, Dr. Drosten and Dr. Malik Peiris embarked
upon different paths of research, only to both arrive at the
same conclusion that the culprit behind the SARS epidemic
was a new kind of coronavirus, one never before known
to infect humans.
Applied Biosystems 3100 System chosen
for critical sequencing steps
Following the RT-PCR procedure, Dr. Drosten's team
determined the nucleotide sequence of amplified DNA
fragments. For this, they used BigDye® terminator sequencing
reagents and a 3100 system from Applied Biosystems to
automate the sequencing reactions5.
It was determined that the SARS-CoV contained nucleotide
sequences different from those found in any other known
coronavirus. In fact, comparative sequence analysis studies
showed that genes from the SARS-CoV shared only a
partial DNA sequence similarity with other known
coronaviruses recorded from previous investigations of
both humans and animals5.
Knowledge of SARS proteins provides clues
for drug researchers
Other SARS-CoV proteins of interest to drug developers
are those that are part of the RNA replication complex
of the virus, enzymes responsible for operating the machinery
that enables the virus to copy its genetic material once
inside a host cell.
Where did SARS come from and where is it going?
To investigate the origins of the SARS virus, scientists
have already studied variations in the amino acid sequence
of components of the spike protein. Or, if they can locate
a similar virus in animals, they may be able to determine
exactly what mutation permitted it to infect humans for
the first time8.
"There are some parallels for coronaviruses in animals,"
notes Dr. Drosten. "For example, there is a kind of pig
coronavirus that once caused very high mortality rates in
pigs, and then it lost some genomic fragment."
A constantly changing genome increases the chances that a
virus will find new ways of infecting individuals which is
frustrating for researchers who attempt to develop a single
vaccine for it. Although there are concerns that as mutations
mount, odds also increase that an even more virulent
SARS-CoV strain will emerge.
If another outbreak of SARS does occur, the rapid response
of research scientists to the initial outbreak of the disease,
coupled with steady advances that have been made in
understanding the nature of the virus will most likely make
it easier in the future to both identify and combat the spread
of the disease.
References
1. Bradsher, K., "Man's Death of 'Bird Flu' in Hong Kong Raises
Fears," The New York Times A7 (February 21, 2003)
2. Pottinger, M., Cherney, E., Naik, G., Waldholz, M., "How Global
Effort Found SARS Virus in Matter of Weeks," The Wall Street
Journal A1 (April 16, 2003)
3. Enserink, M., "The Big Question Now: Will it Be Back,"
Science 301:299 (July 18, 2003).
4. "Abbott Laboratories Enters Agreement with Artus GMBH
for SARS Diagnostic Test," Press Release, Abbott Laboratories,
Abbott Park, Illinois (May 15, 2003)
5. Drosten, C. et al., "Identification of a Novel Coronavirus in Patients
with Severe Acute Respiratory Syndrome," The New England
Journal of Medicine 348(20): 1967-1976 (May 15, 2003).
6. Liu, E., "Science and Societal Challenges: SARS, Singapore, and its
Biomedical Research Institute," presentation at International
Congress of Genetics meeting in Australia (July 10, 2003)
7. Anand, K., et al., "Coronavirus Main Proteinase (3CLpro)
Structure: Basis for Design of Anti-SARS Drugs," Science 300:
1763-1767 (June 13, 2003).
8. Pottinger, M., Regalado, A., "Mutating Virus Keeps Scientists
Chasing SARS," The Wall Street Journal B1 (May 09, 2003)
Contact
us
applications
High-Throughput
gene expression system
for a liver carcinoma research project at CEPH, Paris
group of Inserm scientists at the Centre d'Etude du Polymorphisme Humain (CEPH) in Paris turned
to Applied Biosystems when it needed high throughput equipment to analyse differentially expressed
genes in hepatocellular tumours.
A
Project leader Dr Jessica Zucman-Rossi explained:
“The aim of our research is to understand the molecular
events and characterise the pathways involved with the
development of hepatocellular carcinomas and, from there,
to identify new diagnostic and prognostic markers.
Following the identification of a number of differentially
expressed genes, we use TaqMan® Gene Expression Assays
to analyse their expression in a series of 100 hepatocellular
carcinomas,
benign
tumours
and
corresponding
non-tumour liver tissues.”
Above
Dr. Jessica Zucman-Rossi,
Centre d’Etude du Polymorphisme Humain
(CEPH), Paris
References
1. J Clin Endocrinol Metab. 2004 Mar;89(3):1476-80.
Hepatocyte nuclear factor-1 alpha gene inactivation:
cosegregation between liver adenomatosis and diabetes
phenotypes in two maturity-onset diabetes of the young
(MODY)3 families. Reznik Y, Dao T, Coutant R,
Chiche L, Jeannot E, Clauin S, Rousselot P, Fabre M,
Oberti F, Fatome A, Zucman-Rossi J,
Bellanne-Chantelot C.
2. Pathol Biol (Paris). 2004 Mar;52(2):60-2.
Genetic alterations in hepatocellular adenomas
Zucman-Rossi J. [Article in French]
3. Nat Genet. 2002 Oct;32(2):312-5. Epub 2002 Sep
23. Bi-allelic inactivation of TCF1 in hepatic
adenomas. Bluteau O, Jeannot E, Bioulac-Sage P,
Marques JM, Blanc JF, Bui H, Beaudoin JC, Franco D,
Balabaud C, Laurent-Puig P,
Zucman-Rossi J.
INFO
“It was important for us to find a robust system that could
cope with the high workload this involved and would allow
us to standardise our procedures.
The TaqMan® Low Density Array looked very promising
in both of these respects and we were impressed that
Applied Biosystems offered such a large range of pre-loaded
and validated assays that could be shipped to us
quickly. We have found the quality of the gene
database and annotations to be very high and like the
fact that the human assays can be complemented
with mouse assay sets if required.” Jessica concluded:
TaqMan Gene
Expression Assays
enter: 112
TaqMan Low Density
Array enter: 113
The articles mentioned in
the references enter: 114
Related article can
be found on:
Page 38
“The Applied Biosystems Application Support team helped
us to design the TaqMan Gene Expression Assays for
our tumour series and the experimental protocols to go
with it. They were also on hand to help us validate and
interpret the first results we obtained.”
“The TaqMan Low Density Array looked very promising and we were impressed that Applied Biosystems
offered such a large range of pre-loaded and validated assays that
could be shipped to us quickly”
applications
Proteomics
Solution
in a leading edge
research environment
S
cientists involved in protein expression analysis today
have more and more to deal with due to complex
combinations of basic workflows and massive amounts of
raw data. Increasingly they need better data management and
analytical tools for sample tracking and visualisation of the
meaning behind the raw data. A growing number of scientists
are now turning to Laboratory Information Management Systems
(LIMS) to remove any obstacles in productivity and streamline
their operations.
During the last year, Applied Biosystems Integrated Software Solutions
(ISS) team has been collaborating with the Biomedical Proteomics
Research Group (BPRG) of the Geneva University, lead by Prof. Denis F.
Hochstrasser, and the Proteome Informatics group of the Swiss Institute
of Bioinformatics to provide a LIMS solution for laboratories of the
BPRG and the proteomics platform of the Medical faculty of
Geneva University.
Think with the end in mind…
A number of challenges were faced before delivering a successful LIMS project:
» Ability to build dynamic workflows. In leading edge research
laboratories, the experiments are often designed during the total
experimental process, based on the intermediate results.
» Availability of specialised workflows for Molecular Scanning,
1DE/2DE master gel for best practices collection and ELISA,
immuno blots SELDI when using classical LC and gel-based
protein identification workflows (1D-SDS, 2D PAGE, uni- and
multi- dimensional mLC, MALDI and ESI MS or MS/MS).
» Robust extensible data management system (SQL*LIMS®)
to enable the Proteome Informatics group of the Swiss Institute
of Bioinformatics to support their infrastructure. A high level
of quality in the collected data and very detailed sample tracking
through the analytical process, from preparation to final protein
identification, is mandatory.
» Flexible workflows design procedures to support the Medical
faculty for the Geneva University to provide a service for their
external customers.
Scientists involved in protein expression analysis today have more and more to deal
with due to complex combinations of basic workflows and massive amounts of
raw data. Increasingly they need better data management and analytical tools
for sample tracking and visualisation of the meaning behind the raw data
Figure 1
SQL*LIMS removed
obstacles in productivity
for scientists at BPRG,
in Geneva University
INFO
LIMS in Proteomics
Research enter: 115
LIMS in Genomics
Research enter: 116
Acknowledgements
P. Binz1,2, S.Paesano3,
P. Zanini4, A. Scherl3,
C. Hoogland1,
L. Allard3,
C. Zimmermann3,
O. Carrette3,
J. Sanchez3, S. Borella4,
R. Castelnovo4,
R. Appel1,2,5,
D.F. Hochstrasser2,3,5
The integrated solution
To fulfil the project requirements, Applied Biosystems ISS
team has provided a solution leveraging the Life Science
LIMS application, from the SQL*LIMS family, and the ISS
Proteomics Component suite (see figure 1).
» Operating flexibility is provided by SQL*LIMS, which allows
users to design their own workflows.
» Easy extensibility is supported by an open interface using
built-in APIs and add-on mechanisms. MS instruments and
third-party tools for gel image and protein identification can
be easily integrated allowing direct transfer of real-time,
bi-directional data in or out of the LIMS system.
» Usability is guaranteed by application-specific interfaces for
most of the basic sample operations (aliquoting, spot picking,
MS data loading, protein identification etc.).
» Enhanced data access features are also made available,
providing complete hierarchical overview of studies,
parent-child sample relationships & protein validation results.
LIMS will become the elective source of data for any further investigations
and a milestone into the ‘biological stream’, with sequencing and
genomics for upstream and transcriptomics for downstream
Along with a high level of integration with the latest generation
of MS instruments, including the 4700 Proteomics Analyzer,
and search engines, this solution allows scientists to leverage a
results driven strategy.
Future and Challenges
The BPRG and the proteomics platform of the Medical
faculty of the Geneva University now have an up-to-date
LIMS system for their proteomics laboratories. Their next
challenge is to educate users about the importance of a
LIMS system as a unique repository of their data. LIMS will
become the elective source of data for any further
investigations and a milestone into the ‘biological stream’,
with sequencing and genomics for upstream and
transcriptomics for downstream.
1. Swiss Institute
Bioinformatics
2. Faculty of Medicine,
Geneva University
3. Biomedical
Proteomics Research
Group, Geneva
University
4. Applied Biosystems,
Italy
5. University Hospital,
Geneva
applications
Detecting a
Peptide Biomarker
for Hypertension in Plasma
Peptide Quantitation Using the 4000 Q TRAP™ System
INFO
4000 Q TRAP System
enter: 117
Related article can
be found on:
Page 14
Acknowledgements
Christie Hunter,
Louisette Basa,
Applied Biosystems,
USA
Introduction
he renin-angiotensin system plays an important role
in regulating blood volume, arterial pressure, and
cardiac and vascular function. Management of this
pathway has become very important in the treatment of high
blood pressure and heart failure. Angiotensin II is a small
peptide biomarker that is used to monitor vascular health
at the endocrine level. Angiotensin I is metabolised to
Angiotensin II by the Angiotensin converting enzyme (ACE).
Many treatments for high blood pressure involve prescribing
ACE inhibitors to block the processing of Angiotensin I by
Angiotensin-converting enzyme. This lowers blood pressure by
reducing the circulating levels of Angiotensin II.
Angiotensin II peptide (Sigma) was used to generate a standard
concentration curve using the method described. Rat plasma
(Pel-Freez Biologicals) was prepared using standard organic
precipitation techniques to remove the protein content,
leaving peptides, metabolites, etc. in solution. The plasma
was mixed with acetonitrile in a 1:4 ratio. The supernatant was
dried down, and then re-suspended in 10% acetonitrile / 0.1 %
formic acid.
Experimental
An LC/MS/MS assay was developed to quantify Angiotensin II
peptide levels in plasma using the 4000 Q TRAP system.
The assay was performed using the TURBO V™ Source
at very high LC flow rates (700 µL/min) to maximise
robustness and throughput without sacrificing sensitivity.
Using a 2x50mm C18 column (Targa, Higgins Analytical)
and a short gradient (5-75% acetonitrile in 0.1%
formic acid in two minutes), the total assay time was under five
minutes. The Angiotensin II peptide eluted from
the column in a narrow peak at a retention time of
1.0 minute.
To investigate the optimal level of sensitivity, the assay was also
performed at nanoflow rates (250 nL/min) using the
NanoSpray™ Source. Using a 0.075 x 150 mm C18 column
(Vydac) at 250 nL/min and a slightly longer gradient
(5-75% acetonitrile in 0.1% formic acid in twenty minutes),
the total assay time was 30 minutes. At low flow rates,
the doubly charged precursor ion was predominant so the
MRM transitions 523.82+ ➞ 263.11+ and 523.82+ ➞ 784.61+
were monitored and summed.
T
Because of the high sensitivity and specificity provided
by Multiple Reaction Monitoring (MRM), this MS/MS
triple quadrupole scan mode was used for the assay.
At high flow, two MRM transitions were monitored for
Angiotenisin II (349.83+ ➞ 371.21+ and 349.83+ ➞ 136.11+).
Both Q1 and Q3 were set to transmit a ~1 Da window
centred on the masses specified. Using the Quantitative
Optimisation tool in Analyst® Software 1.4, instrument
parameters were automatically optimised for each
MRM transition.
The identification of the proteins is based on the mascot score
of the peptide mass fingerprint and of the daughter
ion spectra of the tryptic peptides
Results from the MRM assays were automatically processed
using the Quantitation Wizard in the Analyst® Software 1.4.
Summing and integration of the MRM peaks, calculation of the
standard concentration curves and statistics were automatically
performed and the results report generated.
Results
Limit of Quantitation of Angiotensin II at High Flow Rates
A standard curve was run to determine the mass spectrometer
response for this peptide under the assay conditions defined
above. The standard peptide was run sequentially at increasing
concentration. The curve was determined to be linear over
four orders of magnitude (0.160 – 2550 fmole on column,
R= 0.9994, linear regression, 1/x weighting, see inset, figure 1).
The peptide could be accurately detected and quantified down
to 160 amole on column using this assay with a signal to
noise ratio (S/N) of 30:1. The MRM trace for 160 amole of
Angiotensin II peptide on column is shown in figure 1.
Typically, S/N ratios of 10/1 define the lowest limit for accurate
quantitation (LOQ), suggesting that limits down to ~50 amole
on column would be achievable and quantifiable by this
method. It was also determined that summing the two
MRM transitions together improved the S/N ratios obtained
and therefore the limits of quantitation.
Limit of Quantitation of Angiotensin at Low Flow Rates
To assess the levels of detection and quantitation at nanoflow
rates, the standard concentration curve was also measured using
the NanoSpray™ Source at 250 nL/min on the 4000 Q TRAP
system. For this assay, the limit of quantitation of Angiotensin
II peptide was 16 amole on column with a S/N of 13/1
(figure 2). Ten fold greater sensitivity was achieved, but at
longer assay times of 30 minutes.
Figure 1.
Standard concentration
curve of Angiotensin II.
MRM LC trace (sum of
349.83+ ➞ 371.21+ and
349.83+ ➞ 136.11+) shows
the limit of detection
of 160 amole on
column (S/N of 30)
at 700 µL/min.
Inset shows the linear
standard concentration
curve (R=0.9994).
Detection of Endogenous Angiotensin II in Rat Plasma
After the standard curve was generated, a sample of
plasma was run under identical high flow assay conditions.
A strong peak was seen at the same retention time and was
determined to have a concentration on column of 6.6 fmole
(figure 3). This calculates back to be an initial plasma
concentration of 330 amole of Angiotensin II per mL of plasma
(0.35 ng peptide / µL plasma). This peak can be putatively
attributed to the presence of Angiotensin II in rat plasma
because the retention time of the LC peak is similar to the
standard curve and because the two fragment ions monitored
by MRM are co-eluting (figure 3, red and blue trace).
Figure 2.
Standard concentration
curve of Angiotensin II.
MRM LC trace shows
the limit of quantitation
of 16 amole on column
(S/N of 13) at 250 nL/min.
Summary of Quantitation Results
The Multiple Reaction Monitoring experiment of the
4000 Q TRAP system allows for extremely sensitive and
selective detection of specific biomarkers in protein-precipitated
plasma. The efficiency of the TURBO V™ Source at high flow
rates allows for rapid and robust assays to be developed while
retaining high sensitivity. Optimal sensitivity is achieved at
nanoflow rates but at a much lower throughput.
Figure 3.
Endogenous Angiotenisin
II detected in rat plasma.
The equivalent of 20 µL
of protein-precipitated rat
plasma was injected on
column and the area of
the peak compared to the
standard curve in Figure 2.
6.6 fmole of angiotesin II
was detected on column,
which corresponds to a
circulating level of 330
amole Angiotensin II
per µL of plasma.
(MRM transitions shown:
red trace 349.83+ ➞ 371.21+
|and blue trace 349.83+
fi 136.11+).
The unique combination of a triple quadrupole and ion trap
functionality in the 4000 Q TRAP System allows for
confirmatory MS/MS information to be obtained at high
sensitivity during the quantitation assay for higher confidence
results in complex mixtures.
Contact
us
Flow Rate
(µL/min)
Assay
Time (min)
LOQ
(amole)
S/N
Dynamic Range
700
4.5
160
30/1
0.16 to 255 fmole
4 orders
0.250
32
16
13/1
0.016 to 51 fmole
4 orders
Table 1.
Limits of Quantitation
(LOQ) obtained for
Angiotensin II at various
flow rates.
applications
Genome-wide
Expression
Profiling
with the
Applied Biosystems
Expression Array System
INFO
Applied Biosystems
Expression Array System
enter: 118
Acknowledgements
Kelly Li, Irene Lui
and Gary P. Schroth,
Applied Biosystems,
USA
Abstract
xpression profiling1 of over 30,000 genes in
paired normal and breast tumour tissue on the
Applied Biosystems Expression Array System revealed
numerous genes, which were differentially expressed in
primary and metastasis tumours, compared to normal tissue
from the same patient. The study included biological and
technical replicates. Changes in the expression levels were
validated by real-time PCR with TaqMan® probe based assays.
The study demonstrates the use of microarrays for genomewide screening for gene expression, in combination with
quantitative real-time PCR for validating and extending results
for genes of interest.
E
Methods
Two biological and two technical replicates were analysed for
three different disease states. Total RNA from normal, primary
and metastasis tumours of two patients was isolated and labelled
with Digoxigenin using the Applied Biosystems RT-IVT Kit.
The labelled cRNA was hybridised to the Applied Biosystems
Human Genome Survey Micro Array and array duplicates
were processed for each of the three tissues for each of the
two patients. Following hybridisation and washing,
chemiluminescent detection assays were carried out on the
arrays, the resulting signal was read on the Applied Biosystems
Chemiluminescent 1700 Microarray Analyzer.
the low correlation between disease conditions in the same
individual indicates substantial gene expression
changes between disease stages
Results and Discussion
Scatter plots of the technical replicates showed a good
correlation for the same sample of the same patient
(figure 1A). The correlation was reduced when comparing
biological replicates of the same disease status in the two
patients (figure 1B), while the correlation was further reduced
when comparing different disease conditions in the same
patients (figure 1C). While the second result points at
differences in the gene expression of the individuals,
the low correlation between disease conditions in the same
individual indicates substantial gene expression changes
between disease stages.
Using ANOVA test P<0.01 for all 12 arrays identified 2,058
genes with significant differences in gene expression level and
2-D hierarchical clustering analysis was performed using
GeneSpring® software (Silicon Genetics, Redwood City, CA,
USA) (figure 2A). The primary tumour expression profile
from patient 1 resembles more the metastasis pattern,
while that of primary tumour sample from patient 2 looked
more like the expression profile in normal tissue.
The gene annotation database and the PANTHER™
classification system for molecular function and biological
process information, which is an integral part of the Expression
Array System, allowed rapid identification of 200 genes
which are involved in various signal transduction pathways
(Expression profiles in figure 2B). Thirty genes with
characteristic clustering (figure 2C) were validated with
real-time PCR and TaqMan probes.
Figure 1.
Scatter Plots of Replicates.
A.
Technical replicates comparing the same
sample condition from the same patient.
B.
Biological replicates comparing the same
disease condition in two different patients.
C.
Comparison of primary tumour tissue with
metastasis tissue from the same patient.
A group of genes could be determined which were not detected in
normal tissue but which show markedly increased expression in the
cancerous tissues across all four samples for the same disease
condition. Such genes could potentially serve as markers for
changes in disease.
A list of ‘unknown’ genes showed significant changes in
Expression levels (p<0.001), based on expression data from the
Applied Biosystems arrays. PANTHER classification allowed
assigning molecular function and biological processes to many of
these ‘unknown’ genes, based on structural classification of the
unknown genes and comparison of these structural motifs with
those of well-characterised gene products. This approach could be
useful to determine potential markers indicating changes in disease.
Expression changes of 30 genes detected by microarrays were
confirmed by real-time PCR using Gene Expression Assays from
Applied Biosystems across the 3 disease conditions and all
4 replicates. For microarrays, gene expression fold changes were
calculated as a ratio of tumour sample to matched normal tissue
after median global normalisation. For TaqMan data, fold change
was calculated using relative quantitation, or ∆∆Ct (delta delta Ct)
method, where ∆∆Ct = ∆Ct tumour – ∆Ct normal, and ∆Ct is
GAPDH normalised threshold cycle Ct. Fold changes seen by
TaqMan assays and by the Expression Array System are similar.
Conclusion
Statistically significant gene expression changes were identified
when comparing normal versus cancerous breast tissues on the
Applied Biosystems Expression Array System. The utility of the
array system in the identification of potential biomarkers and novel
gene functions using the PANTHER classification system was
demonstrated. TaqMan probe-based assays confirmed expression
changes observed on the microarray. Genome wide expression
screening using the Expression Array System can be easily linked
with quantitative real-time PCR for validating results for genes of
interest. These TaqMan assays can also be used to extend the array
observation across many new samples.
References
1. Genome Wide Expression Profiling of Paired Cancerous and Normal
Breast Tissue. Conference poster by Kelly Li, Irene Lui and Gary P.
Schroth, presented at the 6th Joint Conference of the American
Association of Cancer Research and the Japanese Cancer Association,
January 25 – 29, 2004, in Waikoloa, Hawaii.
Genome wide expression screening using the Expression Array
System can be easily linked with quantitative real-time
PCR for validating results for genes of interest
Figure 2.
2-D hierarchical clustering analysis of expression profiles on the Human Genome Survey Microarray. Red and blue colour represents high and low expression respectively and yellow
represents unchanged expression. All experimental and biological replicates are clustered together. All arrays for normal (N), and metastasis (MT) tissue were clustered into their own
groups, primary tumour (PT) are associated with the pattern of normal or metastasis profiles they resemble more. A) 2,508 genes clustered into two main clusters. The top cluster
indicates increasing expression and the bottom cluster shows decreasing expression of genes in tumours relative to normal tissue. B) Expression patterns of 200 out of 2,508 genes,
which are involved in signal transduction pathways as classified by the PANTHER system. C) Cluster analysis of 30 genes used in TaqMan validation studies.
applications
Assessment of Apoptosis and
lead compound Cytotoxicity
Using the Applied Biosystems 8200 Cellular Detection System
Abstract
poptosis is involved in almost every physiologic
and pathogenic process in the body. Knowledge of the
molecular mechanism of apoptosis has revealed
new approaches for identifying small-molecule drugs that may
effectively treat diseases including cancer, autoimmunity,
stroke, and osteoporosis. In addition, it is important to
determine whether lead compounds are cytotoxic in order to
eliminate them early in the drug discovery process.
A
An apoptosis and a necrosis assay have been developed for use
with the Applied Biosystems 8200 Cellular Detection System
An apoptosis and a necrosis assay have been developed
for use with the Applied Biosystems 8200 Cellular Detection
System. The apoptosis assay uses Annexin V labelled with
a red laser compatible dye to identify apoptotic cells,
while the necrosis assay uses a DNA binding dye that is
excluded from live cells. Both assays utilise CentriRed®
stain for enumeration of the cells present in the scan area.
The 8200 system itself uses a red laser to scan 96-, 384- and
1536-well plates and quantifies cell- or bead-associated
fluorescence using a proprietary algorithm. These assays are
mix-and-read and can be performed on either adherent or
suspension type tissue culture cells.
B
Alexa® 647- Annexin V
1
Annexin V-Alexa®647
2
Annexin V-APC
TOTO®-3
Percent apoptosis/necrosis
A
4
3
TOTO® -3
Introduction
Apoptosis (programmed cell death) is a regulated process
that multicellular organisms use to destroy superfluous cells.
The apoptotic cell bodies are phagocytosed prior to cell
lysis with little or no immune response, thereby eliminating
activation of the detrimental inflammatory process.
Apoptosis involves two known pathways that can be termed
extrinsic and intrinsic. The extrinsic pathway is stimulated by
protein or small molecule ligands that bind to cell surface death
receptors (e.g. FasL) and activate the upstream caspases directly.
The intrinsic pathway is induced by cellular stresses and refers
to mitochondrial processes that involve the BCL-2 family of
proteins, causing the release of cytochrome C and leading to the
activation of downstream caspases1. Both pathways converge
with the activation of caspase-3 and induce morphological
changes such as cytoplasmic shrinkage, active membrane
blebbing and chromatin condensation. Biochemical changes
include the activation of caspases, the externalisation of
phosphatidylserine and DNA fragmentation. Annexin V
binding to phosphatidylserine exposed to the outer leaflet of the
plasma membrane has been shown to be a reliable early marker
for cells undergoing apoptosis2. Recombinant Annexin V
labelled with a red laser dye and CentriRed stain have been
combined to generate a 2-colour apoptosis assay that can be
performed in 96- or 384-well plates (figure 1A). We have used
human hepatocellular carcinoma (HCT116) cells treated with
staurosporine as a model system for apoptosis. Staurosporine is
a potent inhibitor of protein kinase C that induces apoptosis in
cultured cells. CentriRed nucleic acid binding dye stains both
viable and dead cells. It is used to enumerate all of the cells in
the scan area so that the data can be presented as the percentage
of cells within the scan area that are binding Annexin V.
Alternatively, cells that undergo necrosis as a response to the
accumulation of toxic compounds display rapid degradation,
increases in plasma membrane permeability, swelling of the
mitochondria and a subsequent host immune response.
TOTO®-3 dye is a high affinity DNA labelling red laser
dye suitable for use on the 8200 system that stains only dead
cells with compromised plasma membranes. In this assay,
TOTO-3 dye is used to identify necrotic cells and CentriRed
dye is used to enumerate all cells in the scan area and the result
is presented as a percentage of the total.
Materials and Methods
Cell culture and Induction of Apoptosis: HCT-116 cells
(Colorectal Carcinoma, ATCC) were plated in 100 µl
complete media (RPMI-1640, 10% FBS and 1% Penicillin/
Streptomycin) at 10,000 cells per well in a 96-well FMAT®
plate. The cells were plated 24 hours prior to drug treatment
to allow them to attach firmly to the bottom of the well.
A dose response curve for staurosporine (Sigma) treatment
was performed.
First
Staurosporine (214 µM in absolute ethanol) was diluted
to a final concentration of 6 µM (6000 nM) in complete
media.
Second
10 serial 3-fold dilutions were performed to produce
all 11 points on the curve with the last column containing
no staurosporine as a control.
Third
100 µl of media or media plus staurosporine was added
to each well (6 replicates) and the final volume in
every assay well contained 200 µl.
NOTE: For the hit plate assay, 6 individual wells were
treated with 3 µM staurosporine and the remaining wells were
untreated. Cells were incubated overnight (18-20 hours) in a
37°C, CO2 incubator
Above: Figure 1.
A) Greyscale images
captured with an 8200
system assay. In the
healthy control wells,
CentriRed stain binding
is visible (images 1 and
3). In the sample wells
undergoing apoptosis,
the characteristic
blebbing can be
visualised in the image
(image 2). In wells
showing necrosis,
the cells stain
predominantly with
TOTO-3 (image 4).
CentriRed stain does
not bind to apoptotic/
necrotic cells that have
lost their nucleic acids
into the surrounding
media. Therefore, in the
wells treated with 3 µM
staurosporine (wells 2
and 4), the binding
is represented by
Dye-labelled Annexin V2
or TOTO-34 only.
B) Graphs showing the
percentage of apoptosis
or necrosis versus the
concentration of
staurosporine (nM).
The percentage is
calculated by dividing
Annexin V or TOTO-3
binding events by
the total number of
events. This assay was
performed in a 96-well
plate and each condition
had replicates of 6.
The estimated IC50 is
35 nM staurosporine
for Annexin V binding
and 1 mM for TOTO-3
dye binding.
Apoptosis (programmed cell death) is a regulated
One hallmark of necrosis is the ability of certain dyes such as
trypan blue and propidium iodide to pass through the
membrane and stain the cell.
process that multicellular organisms
use to destroy superfluous cells
Page 30
Note: For the hit plate assay, 6 individual wells were treated with
3 mM
A staurosporine and the remaining wells were untreated.
Cells were incubated overnight (18-20 hours) in a 37°C,
CO2 incubator.
B
Left: Figure 2.
Staurosporine-induced
apoptosis measured
with fluorescent
dye-labelled Annexin V
to illustrate a screening
assay.
A) Bar graph of
apoptosis data from a
96-well FMAT® plate
of HCT116 cells
treated with 3 µM
staurosporine in wells
2a, 3e, 6d,9c, 9f, 10a
and 11c.
B) 3D graph illustrating
hit analysis using the
8200 analysis software
to plot cell count
for Population A
(Annexin V-Alexa
Fluor®647 binding
events).
C
Red laser dye-labelled Annexin V binding assay: CentriRed
stain (Applied Biosystems: 10 µM stock in anhydrous
methanol) and Annexin V-Alexa Fluor® 647 (Molecular
Probes) were added to 5x Annexin V binding buffer (12.5 mM
Calcium Chloride, 140 mM Sodium Chloride, 10 mM Hepes)
at concentrations of 15 nM and 1:800 respectively. 50 µl of
the above dye mixture was added to each well of the
staurosporine-treated 96-well plate so that the final volume was
250 µl. The addition of both CentriRed stain (final 3 nM)
and Annexin V-Alexa Fluor 647 (final 1:4000) allows 2-colour
analysis and calculation of the percentage of cells in the scan
area that are binding to the Annexin V protein. For the
Annexin V-APC assay, Annexin V-APC (Molecular Probes)
replaced Annexin V-Alexa Fluor 647 at a final concentration
of 1:4000.
TOTO-3 dye binding assay: CentriRed stain (Applied
Biosystems) and TOTO-3 dye (Molecular Probes) were
added to 5x Annexin V binding buffer at final
concentrations of 15 nM and 75 nM respectively.
50 µl of the above dye mixture was added to each
well so that the final volume was 250 µl. The addition
of both CentriRed stain (final 3 nM) and TOTO-3 dye
(final 15 nM) allows 2-colour analysis and calculation
of the percentage of cells in the scan area that are stained
with TOTO-3 dye. For both assays the plates were
incubated in the dark at room temperature for 1-4 hours
prior to scanning.
C) Well detail of
6c and 7c to show
pseudocolour images
and colour gating.
In these examples,
Annexin V-Alexa
Fluor®647 (Dye A)
is represented by blue
images and CentriRed
stain (Dye B) is
represented by
pink images.
INFO
Results and Conclusions
It is believed that the ability of chemotherapeutic drugs to
induce apoptosis increases their efficacy3. Therefore, the ability
to rapidly screen synthetic or natural compound libraries for
small molecule drugs that induce apoptosis is useful for drug
discovery efforts in oncology. In addition, defects in the
apoptotic pathway may be involved in other disorders
associated with cell accumulation such as autoimmunity and
inflammation. The 8200 system apoptosis assay is based on the
binding of dye-labelled Annexin V to phosphatidylserine that
is exposed to the outer surface of the cell membrane in cells
undergoing apoptosis. The IC50 for staurosporine-induced
apoptosis in this assay has been determined to be
35 nM which is consistent with literature values. This assay has
been shown to be robust and reproducible. We also show that
TOTO-3 dye can be used in the 8200 system to measure the
amount of necrotic cells present in an assay well. The IC50 for
staurosporine-induced necrosis has been determined to be
1 mM. This assay can be used in addition to the Annexin V
binding assay to gain more information about whether the test
compound also causes necrotic cell death. In addition to the
numerical data reported, grey scale and pseudocolour
images are generated that enable well-by-well analysis of
hits to observe the state of the cells that are identified as
apoptotic or necrotic.
The 8200 system is an excellent tool for quantitative analysis
of fluorescent molecules bound to the surface of tissue culture
cells because they have minimal autofluorescence from the 633
HeNe laser4. The 8200 system uses macroconfocal technology
to collect data from a 1mm2 area on the bottom of the
well with an approximately 100 micron depth of focus.
Cell-associated fluorescence is collected by two PMTs (PMT1,
650-685; PMT2, 685-720 nm) and allows the deconvolution
of two different red laser dyes run concurrently in mix-andread format. Multiple 96-, 384- or 1536-well plates can be
loaded onto the automated plate handler allowing analysis of
thousands of compounds per day. Cell-based receptor-ligand
binding and hybridoma screening assays, and bead-based
immunoassays can also be performed on the 8200 system.
References
1. Reed J.C.and Tomaselli, K.J. Drug discovery opportunities from
apoptosis research. Curr Opin Biotechnology, 2000 6:586-92.
Applied Biosystems
8200 Cellular Detection
System enter: 119
2. Van Engeland, M., Nieland, L.J., Ramakers, F.C., Shutte, B.,
Reutelingsperger, C.P. Annexin V-affinity assay: a review on an
apoptosis detection system based on phosphatidylserine exposure.
Cytometry 1998; 31(1):1-9.
3. Tamm I., Schriever, F., Dorken, B. Apoptosis: implications of basic
research for clinical oncology. The Lancet-Oncology 2001; Vol 2:33-42.
4. Lee J.Y., Miraglia S, Yan X, Swartzman E, Cornell-Kennon S,
Mellentin-Michelotti, J, Bruseo, C, France D.S. Oncology Drug
Discovery Applications Using the FMAT® 8100 HTS System.
J Biomol Screen. 2003 Feb;8(1):81-8.
Acknowledgements
Julia M. Michelotti,
Lolita Evangelista,
Applied Biosystems,
Foster City, USA
Carol Khodier,
Sonia Connaughton,
Applied Biosystems,
Bedford, USA
Contact
us
product news
New
BioTrekker
Software v1.0
™
Offers a solution to your genotyping data management problems
INFO
BioTrekker Software v1.0
enter: 120
L
arge genotyping projects generate a great quantity of
data, which need to be managed efficiently to allow
rapid and accurate derivation of final linkage or
association results. Some considerations involved in this
task are the links between subject details and the relevant
genotype data, the performance of quality control and data
concordance tests, and the capability to archive and export
finished data files in a suitable format for downstream
linkage or association analyses.
Further benefits come from the ability to import genotype
data from different analysis platforms and assay technologies,
to draw together all available experimental results into
one database.
Acknowledgements
Dave Watts,
Applied Biosystems, UK
The new BioTrekker Software v1.0 from Applied Biosystems
addresses the need to provide a complete solution for genotyping
data management. It allows you to connect to GeneMapper®
Software v3.5 and Sequence Detection System Software v2.2
Enterprise database to import analysed genotyping data.
» The GeneMapper Software v3.5
data analysis from the SNPlex™
and microsatellite assays, run on
3730, 3730xl, 3100 and
electrophoresis platforms.
provides upstream raw
system, SNaPshot® kit
the Applied Biosystems
3100-Avant capillary
» The SDS Enterprise software analyses data from the
ABI PRISM® 7900HT Sequence Detection System, running
allelic discrimination assays for SNP genotyping including
the comprehensive range of Taqman® SNP genotyping
assays from Applied Biosystems.
BioTrekker Software v1.0 offers flexibility in installation:
it can be installed into a GeneMapper v3.5 database instance
on an existing dedicated GeneMapper workstation, or run
as a stand-alone system with its own Oracle 8.1.7 Standard
Edition database. As a stand-alone system, it uses the
same computer specification level as the separate workstation
for GeneMapper Software v3.5, which is equivalent to that
supplied with our current multi-capillary genetic analysis
instruments (BioTrekker software cannot be supported on
Capillary Electrophoresis instrument Data Collection or
Enterprise workstations).
The genotyping database manager allows all typing
results to be collated in one central database and can
create and manage consensus genotypes and perform
concordance tests. Quality control tests can be carried out
on datasets, with the ability to query and view individual
genotype results when necessary. All valid data can be exported
to text files, enabling downstream statistical analysis to
establish linkage or association results.
Finally, all data can be safely archived, and BioTrekker
Software’s security features control database access and can
assist in meeting 21 CFR 11 compliance. BioTrekker Software
v1.0 offers the scientist running a genotyping project the
means to take control of all their data management.
It allows you to connect to GeneMapper Software v3.5
and SDS v2.2 Enterprise database to import
analysed genotyping data
Contact
us
product news
NanoMate
®
100 System
For the QSTAR®, API 3000™, API 4000™ & 4000 Q TRAP™ Systems
T
The ESI Chip
The disposable ESI Chip used with the NanoMate 100 is a
microarray of 100 nanoelectrospray nozzles etched in a silicon
wafer, to facilitate stable nanoelectrospray at low flow rates
(figure 1). Each nozzle is made to exactly the same
specification so that every nanoelectrospray is identical
and provides fully reproducible spray. The ESI chip provides
a novel, reliable, and reproducible electrospray of fluid
samples for MS analysis.
he NanoMate 100 with ESI Chip™ from Advion
BioSciences (Ithaca, NY, USA) is a fully-automated
chip-based nanoelectrospray system suitable for
our high-end API mass spectrometers. The NanoMate
combines automation and miniaturisation to increase sample
throughput, sensitivity and information content from trace
biological samples.
The NanoMate combines automated sample handling with
the technology of the ESI Chip to produce data from hundreds
of samples per day. To analyse samples, the NanoMate uses a
conductive pipette tip to draw sample from a 96 well-plate.
The sample-filled tip aligns with a nozzle inlet on the back of
the disposable ESI Chip, creating a tight seal. Each pipette tip
and nozzle is used only once, providing a unique path into
the mass spectrometer and eliminating sample carryover.
Following nanoelectrospray and MS analysis, the NanoMate
can return unused sample to the well or pick up a new sample
and begin the process again.
Figure 1.
The
ESI Chip
The NanoMate combines automation and miniaturisation to
increase sample throughput, sensitivity and information
content from trace biological samples
Metabolite Identification
To illustrate the capabilities of the NanoMate for metabolite
identification, erythromycin was analysed for metabolites
without LC separation using the source on a QSTAR® Pulsar
Hybrid LC/MS/MS System in full MS/MS and precursor
ion scanning modes. Erythromycin was incubated at 10 µM
o
with pooled human microsomes for 1 hr at 37 C to generate
metabolites. Sample preparation consisted of adding
acetonitrile to the sample (1:1) and centrifuging for
2 minutes at 8,000 rpm. The supernatant was filtered
with a 0.2 µm filter. Both LC/MS, using an ion spray source,
and NanoMate/MS analysed the same samples for direct
comparisons. Samples were desalted using Millipore ZipTips®
prior to infusion analysis on the NanoMate.
Figure 2 shows spectra from the NanoMate and LC/MS
runs for an incubated sample of erythromycin. Both spectra
were expanded around the mass range of interest to
illustrate the parent and metabolites. This figure clearly shows
that the NanoMate and LC/MS data were comparable.
Furthermore the infusion analyses offered by the NanoMate
provide the necessary extended analysis time for precursor ion
scanning. Figure 3 shows results of precursor ion scanning with
the NanoMate at a step size of 1.0 amu (top) and 0.1 amu
(bottom). The infusion analysis permits the user to acquire
many more data points and thus achieve better data quality
for precursor ion scanning.
Figure 2.
NanoMate (top) and
LC/MS (bottom):
Erythromycin “Full Scan”
TOF (Expanded region
showing metabolites).
The mass spectrum from
the LC/MS data shown
is the average of the LC
elution window from
2 to 3 minutes
Mapping Protein Phosphorylation Sites
on a QSTAR System
The extended infusion time obtainable with the NanoMate
provides added flexibility for post-translational mapping
studies. In this example, a standard bovine β-casein tryptic
digest was analysed using a precursor ion scan of m/z 79
in negative ionisation mode. Once the phosphopeptides
were identified, MS/MS in positive ion was performed in
order to sequence the peptides. The results showed that all
phosphorylation sites of β-casein tryptic digest could be
mapped at 50 fmol/mL. Figure 4 shows the MS/MS spectrum
obtained from the phosphopeptide parent ion m/z 1031.4.
These results demonstrate that the automated chip-based
nanoelectrospray platform is a valuable system for
phosphorylation analyses due to stable, extended infusion
times for completing precursor ion scanning followed by
MS/MS on the identified phosphorylated peptides.
Figure 3.
Results from precursor
ion scanning with the
NanoMate. The precursors
of m/z 158 of erythromycin
incubate were investigated
using a step size of 1.0 amu
and 0.1 amu.
Figure 4.
Positive ion MS/MS
spectrum of m/z 1031.4
from 50 fmol/mL of bovine
β-casein tryptic digest.
The spectrum
unambiguously
identifies the site of
serine phosphorylation
Instrument Compatibility
Advion has designed brackets to mount the NanoMate 100
to the QSTAR, API 3000, API 4000 and 4000 Q TRAP
LC/MS/MS systems. Applied Biosystems and Advion, via their
strategic alliance, both ensure system compatibility between
the NanoMate 100 and these LC/MS/MS platforms.
Conclusion
The Advion NanoMate 100 robotic system further enhances
the versatility and performance of the Applied Biosystems
LC/MS/MS systems. In combination with the disposable
ESI Chip, the NanoMate allows users to take full advantage of
the capabilities of nanoelectrospray mass spectrometry for life
science applications, notably by providing extended acquisition
time for proteomics and metabolite identification applications
where greater sample information is required. Key advantages
include higher sample throughput, low sample consumption,
high sensitivity, reduced analysis cost and the ability to derive
meaningful information from a very small sample.
INFO
please contact your local
Applied Biosystems
Sales Engineer.
Acknowledgements
Jean-François Alary,
MDS-Sciex,
Canada
Colleen Van Pelt,
Advion BioSciences Inc.,
USA
The ESI chip provides a novel, reliable,
and reproducible electrospray of
Contact
us
Whether you have a question, query or comment on this article, or any of the others
in this issue, please get in touch with us at: [email protected]
fluid samples for MS analysis
product news
New Amine Specific
iTRAQ™ Reagents
expand multiplexing
and quantitation
capabilities for
Proteomic researchers
INFO
iTRAQ Reagents
enter: 121
or contact your local
Applied Biosystems
Sales Engineer
Background
s research moves more towards understanding
protein function, studying protein-protein interactions,
and monitoring changes in protein profiles
from systematic challenges, there is a need to develop
quantitative tools. Recently, there has been an increasing
desire to simultaneously compare multiple samples in a
relative or absolute manner and a need to develop tags that
provide broad proteome coverage while maintaining
important structural information such as post-translational
modifications. The requirements have led to the development
of the iTRAQ reagents.
A
iTRAQ Reagents
Applied Biosystems iTRAQ reagents are a multiplexed
set of non-polymeric, isobaric reagents which yield
amine-derivatised peptides from a protein digest, that are
chromatographically identical and indistinguishable in MS,
but produce strong low-mass MS/MS signature ions that
permit quantitation. This tagging system allows you to extract
more detailed information from samples because important
information, such as post-translational modifications, is not
lost in the process.
giving the statistical relevance needed for quantitative
experiments. We have incorporated this quantitation
technology into a simple workflow of labelling the resulting
peptides after parallel protein extraction and routine
enzymatic digestion common to any LC/MS experimental
procedure. The use of these labels therefore permits
simultaneous measurement of relative and/or absolute
protein abundance of multiple, samples in a single
LC/MS/MS run.
Key features and benefits
» Expanded proteome coverage by labelling all peptides,
including those with post-translational modifications
(PTMs) to extract more detailed information from
your samples.
» Enhanced low-level analysis as a result of the signal
amplification from the additive fragmentation of
labelled isobaric peptides resulting in greater depth into
sample information
» Increased confidence in identification and quantitation by
Acknowledgements
Lynn Zieske,
Applied Biosystems,
Foster City, USA.
Tony Hunt,
Applied Biosystems,
Framingham, USA
Since all peptides are tagged, proteome coverage is expanded
and analysis of multiple peptides per protein improves
the confidence in those identified. Protein identification is
simplified by improved fragmentation patterns, with no signal
splitting in either the MS or MS/MS modes and the
complexity of data is not increased by mixing multiple samples
together. Additionally, the ability to simultaneously analyse up
to four different samples enables the comparison of numerous
sample states and provides the flexibility to
include duplicates or triplicates into experimental design,
Expanded proteome coverage by labelling all peptides, including
those with post-translational modifications (PTMs) to extract
more detailed information from your samples
tagging multiple peptides per protein to gain more
statistically significant information.
» Analysis of up to four different biological samples
simultaneously in a single experiment.
» Perform absolute quantitation across numerous sample
states, for the synchronous uniform comparison of normal,
diseased and/or drug treated states.
» Simple workflow labels peptides allowing rapid progression
to LC/MS/MS analysis and easy data interpretation
with Pro QUANT Software for relative and absolute
quantitation.
The iTRAQ Reagent structure
Comprises of two main segments:
1. Peptide Reactive Group (PRG) – Covalently links the
iTRAQ reagent to the free primary amines (N-terminal
amines and lysine side chains) in the peptide.
2. Isobaric Tag – Isobaric, by definition, implies that any two
or more species have the same atomic mass but different
arrangements. Choosing an isobaric tagging system
enables multiple samples to be tagged with no resulting
increase in MS complexity when quantitation occurs upon
fragmentation in MS/MS space. As illustrated in figure 1,
the iTRAQ reagent tag consists of reporter group (tag after
MS/MS fragmentation) and balance group (to keep the
overall isobaric tag mass the same for each reagent.
The balance portion of this reagent is ultimately invisible
upon fragmentation at the MS/MS Fragmentation Site.
During MS/MS, fragmentation occurs on both sides of the
balance group, resulting in neutral loss of the balance group
and releasing the reporter group. The reporter groups
appear in the low mass region between m/z 113-119,
an area specifically selected because no common fragment
ions have been found to appear in that region.
Isobaric tagging of peptides for multiplex analysis
A key feature of the iTRAQ reagents is, when combined,
the MS of the iTRAQ Reagent-labelled sample digest
mixture resembles the MS of an individual sample (assuming
the same peptides are present). The balance group ensures
that an iTRAQ reagent-labelled peptide has the same m/z,
whether labelled with iTRAQ Reagent 114, 115, 116, or 117.
In the example illustrated, a six protein mix was labelled
5:1:10:1 with iTRAQ reagents 114, 115, 116 and 117,
respectively. The samples were mixed and the resulting
MS and MS/MS spectra recorded using a QSTAR® XL system.
As seen, there is no increase in complexity in the overall spectra.
There is a single parent ion in the MS spectrum (see figure 2
inset), cumulative from all four samples. In addition, the y- and
b- ion rich MS/MS spectra is also not complicated by multiple
signals except in the important reporter region in which the
relative ratios of the three species are recorded.
Absolute quantitation
Quantitation of specific proteins of interest can also be
performed using the iTRAQ reagents. This involves
comparing peptides of interest of the targeted protein(s)
to known (quantified) amounts of labelled synthetic standard
peptides, representing unique fragments of the protein(s)
of interest, spiked into the sample. By labelling the spiked
in peptide with one of the iTRAQ reagents and the
sample(s) labelled with one or more of the other reagents,
both relative and absolute quantitation can be obtained in a
single MS/MS spectrum.
= Fragmentation site
Analysis of up to four different biological
Above. Figure 1.
samples simultaneously in a
single experiment
VLVDTDYK
Software for data analysis
The Pro QUANT software package supporting the iTRAQ
Reagents is available for the QSTAR® XL and Q TRAP® family
of MS systems.
Conclusions
Whether you are performing protein expression analysis
or absolute quantitation experiments, Applied Biosystems
iTRAQ reagents coupled with our MS instrumentation
provide the features, the confidence, and the statistical
relevance you need for quantitative biology. In addition
to PTM analysis, these reagents are also ideal for
laboratories performing discovery/validation analyses and
time course studies.
Above. Figure 2.
QSTAR XL system
Q TRAP system
Contact
us
product news
Applied Biosystems LIMS
for the Life Sciences
Transforms the way your
laboratory operates
INFO
F
or most high-throughput biological laboratories, an
increase in throughput means an increase in operating
complexity. Managing an ever more complicated
workflow has become vital for a laboratory to increase
productivity, improve data quality, reduce costs, and integrate data
from many different sources.
Recently released, the Applied Biosystems LIMS is a
revolutionary new workflow management, process automation,
and data integration solution for the high-throughput
biological laboratory.
Visit:
www.appliedbiosystems.
com/InformDisc
or email:
InformaticsInfo@eur.
appliedbiosystems.com
Applied Biosystems LIMS includes a comprehensive set of features
to help you integrate, automate, and manage all your data and
workflow processes. The software is built on the fully scalable,
extensible, multi-tiered architecture from Applied Biosystems and
leverages the core sample management functionality found in their
award-winning SQL*LIMS® Software. This software is the
Laboratory Information Management System (LIMS) of choice
for more than 30,000 professionals in 1,000 laboratories across a
wide range of industries.
Related articles can
be found on:
Pages 21, 26 & 50
The features of Applied Biosystems LIMS allow you to:
Manage samples and containers and organise data
» Map sample transfer and re-array from container(s) to
container(s) using a flexible container mapping tool
Acknowledgements
Maria Rodrigues,
Lori Graham,
Applied Biosystems,
USA
» Track samples and containers through their life cycles
» Organise and track experiment and study data with user-defined
project folders
a revolutionary new workflow management, process automation,
and data integration solution for the high-throughput
biological laboratory
Figure 1.
Drag-and-drop
graphical workflow
editor to define
end-to-end
laboratory
workflow process
Support a changing laboratory operation
with flexible, adaptable features
» Easy-to-use, drag-and-drop, graphical workflow editor
»
»
»
»
Reusable activities, protocols and workflows
True workflow engine for automatic job queue and data flow
Fully documented API and developer tool kit
Library of specific Applied Biosystems and third-party
instrument interfaces
» Result parser for importing results from
third-party instruments
» Out-of-the-box workflow to support Applied Biosystems
Taqman® Gene Expression, SNP Genotyping Assays and
SNPlex system
» Pluggable user interface in activity-driven laboratory console
Figure 2.
Activity-driven Laboratory Console to view and manage
daily laboratory tasks and work lists.
Maximise laboratory process and research efficiency
» Track consumables and sample inventory
» Monitor status of ongoing laboratory operations and
instruments using the process viewer
» Promptly notify users of process errors using user-defined
notification feature
» Manage assays by markers and track assay reagents
Support data security and assist with
21 CFR Part 11 compliance
» Control access by data group and jobtype functions with
user-configurable feature
» Get user-configurable audit trail and electronic signature
Applied Biosystems LIMS is truly the next generation laboratory
management and automation software. It is designed to transform
the way your laboratory operates, helping you integrate,
automate and manage all your laboratory data and workflow
processes. Informatics systems from Applied Biosystems
are backed by our global support, professional services,
and Informatics Specialists worldwide.
product news
Announcing
Real-Time
the family of
PCR platforms from Applied Biosystems
– instruments to suit all requirements
INFO
S
Applied Biosystems 7300
Real-Time PCR System
enter: 122
enter: 123
ABI PRISM 7900HT
Sequence Detection
System enter: 124
Acknowledgement
Falko Kraeusche,
Applied Biosystems,
Germany
ince Applied Biosystems pioneered real-time PCR in 1995, we have continued to develop the
technology to provide more powerful solutions for labs of all sizes. With the introduction of the
Applied Biosystems 7300 and 7500 Real-Time PCR Systems, we can now offer three different
instruments for all possible requirements and applications.
Which instrument is best suited to your requirements?
The Applied Biosystems 7300 Real-Time PCR System is the
perfect entry to real-time PCR, producing high-end results
within budget. The System uses a standard 96-well format.
The filter wheel with four emission filters allows the detection
of all dyes between 510nm and 650nm. Through an advanced
multicolour detection algorithm the system can analyse up to
4 dyes simultaneously and enables you to perform a wide
variety of applications – including gene expression analysis,
pathogen quantification, SNP genotyping and +/- assays to
confirm the presence or absence of specific targets.
For all customers who need to use a broader range of
fluorophores, the Applied Biosystems 7500 Real-Time PCR
System is the right choice. The system has an advanced optic
unit with two 5-filter wheels, which give a higher sensitivity
especially for redder dyes. The Relative Quantitation (RQ)
Study software is a standard component of the 7500 system,
while it is optional for the 7300 instrument. The RQ Study
software allows you to generate gene expression profiles from
up to ten 96-well plates in seconds. The really revolutionary
characteristic, however, is the new high-speed thermal cycling
upgrade option. All users who need real-time PCR results
quickly can obtain them in less than 40 minutes, in a single
tube format or using a 96-well plate. The 7500 system offers
additional performance across the same range of applications
as the 7300 system.
With the introduction of the Applied Biosystems 7300 and 7500
Real-Time PCR Systems, we can now offer three different
instruments for all possible requirements and applications
The ABI PRISM® 7900HT Sequence Detection System
completes the series of real-time PCR instruments from
Applied Biosystems. The system offers unmatched flexibility
and throughput with user interchangeable thermal cycling
blocks and an optional Automation Accessory for automatic
plate loading and unloading for true walkaway automation.
Currently 384-well, 96-well or TaqMan® Low Density Array
thermal cycling blocks are available. The TaqMan Low Density
Array is a custom-configured 384-well low volume micro
fluidic card (2µL reaction volume per well) which allows
low-density gene expression array experiments to be performed
without the need for sample handling robotics. TaqMan® Gene
Expression Assays are pre-deposited and dried down into the
reaction wells during the manufacturing process, leaving only
a simple sample loading process to be performed by the system
user. The result is an extremely easy-to-use low density
array with the gold standard data quality provided by
Applied Biosystems TaqMan Gene Expression Assays and
real-time PCR systems.
The TaqMan Low Density Array can accommodate between
12 and 380 gene targets, up to 8 samples per array, and offers
three choices of replicate numbers. The 7900HT system also
offers a comprehensive software suite which simplifies
high-throughput data analysis and provides a comprehensive
set of features for users working in a validated environment.
The ABI PRISM 7900HT Sequence Detection System is
Applied Biosystems flagship real-time PCR system, and is
the most capable, most flexible and highest throughput
system available from Applied Biosystems.
ABI PRISM 7900HT
Sequence Detection System
Block Format
Fixed 96-well
Fixed 96-well
User interchangeable 96-well, 384-well
and TaqMan Low Density Array*
Support for
TaqMan Low
Density Array
No
No
Yes
Excitation Source
Halogen lamp, fixed wavelength
Halogen lamp, variable wavelength
Laser
Emission
4 colour detection
5 colour detection
Continuous wavelength detection
from 500-660nm allows highly flexible
multi-colour detection capabilities
Future upgrade to
fast thermal
cycling
No
Yes
Yes
Automated Plate
Loading/Unloading
No
No
Yes, with optional Automation Accessory
Software
Applications/Assay
Types
Absolute quantitation (standard curves),
Allelic Discrimination (SNP genotyping)
and Plus/Minus assays using an
Internal Positive Control
Relative Quantitation Study
(comparative CT method), Allelic
Discrimination (SNP genotyping) and
Plus/Minus assays using an Internal
Positive Control
Relative Quantitation Study (comparative
CT method), Allelic Discrimination
(SNP genotyping)
Automatic CT
determined
Yes
Yes
Yes
Automated Allele
Calling
Yes
Yes
Yes
Optional Software
Relative Quantitation Study
(comparative CT method)
Software features
and functions to
support customers
requiring validation
No
No
Yes, software includes functions and
features to support customers requiring
21CFR part 11 compliance
Includes Primer
Express™ Software
Yes
Yes
Yes
Summary
Full-featured instrument that provides
high-end results within budget
Additional performance and features in
the 96-well format
Most capable, flexible and highthroughput real-time PCR instrument
Enterprise database software to support
high-throughput gene expression and
SNP data collection and analysis
* TaqMan Low Density Array requires an initial service upgrade, after which the thermal cycling block is user interchangeable
Depending on your application, throughput needs and
budget you can now choose between three real-time PCR
instruments from Applied Biosystems. In an integrated science
approach, the real-time PCR instruments combined with
our high-quality sample preparation, optimised real-time
PCR chemistry, validated TaqMan Gene Expression Assays or
Custom TaqMan Gene Expression Assays, these instruments offer
you a complete solution for your Real-Time PCR experiments.
This gives you reliable results easier and faster.
Researchers in genomics can speed up their projects still
further by taking advantage of rich genomic information
provided in the myScience research environment:
http://myscience.appliedbiosystems.com
the PANTHER™ Classification System for molecular function
and biological processes, or the subscription-based Celera
Discovery System™ Online Platform.
product news
SQL*LIMS
®
v5 Software
The best in LIMS just got better
INFO
visit: www.sqllims.com
Or email:
InformaticsInfo@eur.
Acknowledgements
Lori Graham,
Vince Woodall,
Applied Biosystems, USA
Peter Boogaard,
Applied Biosystems,
The Netherlands
A
pplied Biosystems announces SQL*LIMS v5
software, an enhanced version of this awardwinning Laboratory Information Management
Systems (LIMS). This new version is the first fully web
enabled LIMS and is a complete enterprise-accessible
solution for pharmaceutical and other QA/QC
manufacturing operations. This new system’s extended
power simplifies large, multi-site deployments;
reduces personnel overhead costs; and provides new,
easy-to-use, streamlined navigation.
First Fully Web-enabled LIMS
SQL*LIMS v5 software’s new, open Web services platform
has been built using industry standard technology
including the Oracle® Application Server 10g and
Database 9i, and supports the extensible Java 2 Platform,
Enterprise Edition (J2EE) standards-based application.
This powerful platform allows you to build and extend
functionality to meet your complete needs for e-commerce
and web messaging.
Further, typical LIMS applications require you to install
software on local user workstations, making the systems more
costly to implement and maintain. With the introduction of
the Web-enabled SQL*LIMS v5, you can reduce your costs
with a more lightweight, configurable system that is easy to
distribute, maintain, and connect to other critical applications.
You also get full Web access from inside or outside your
firewall. And you can do all this while still securely protecting
your data quality and integrity.
Reduce Your Costs
A comprehensive LIMS is critical for today’s QA/QC
manufacturing organisations to maximise automation,
optimise laboratory processes, and create a truly paperless
laboratory. Consequently, the return on investment of a
LIMS is usually significant, with a quality system reducing
staff errors, identifying process bottlenecks, increasing
analysis accuracy, and reducing material and reagent waste.
A LIMS can also potentially reduce your project
turnaround time from years to months.
New Expanded Features
Applied Biosystems greatly extends the power of earlier releases
with major new features and functionality in SQL*LIMS v5.
The new software can be easily configured to meets both the
requirements of a small laboratory or large, global operation.
New expanded features include:
» Instrument calibration management
» Analyst training and qualification tracking
» Reagent inventory tracking
» Streamlined, two-mouse click navigation
» Enhanced fill-down, pop-ups for data entry and approvals
» Secure single user password for users of multiple
database instances
This new version is the first fully web enabled LIMS and is a complete enterprise-accessible solution for pharmaceutical and
other QA/QC manufacturing operations. This new system’s extended power simplifies large, multi-site deployments;
reduces personnel overhead costs; and provides new, easy-to-use, streamlined navigation
Proven Quality and Performance, Fully Validated
SQL*LIMS Software is the industry’s most innovative, end-to-end
solution to integrate, automate, and manage laboratory data and
workflow processes. That is why it is the LIMS of choice for
more than 30,000 professionals in 1,000 laboratories across a wide
range of industries.
SQL*LIMS v5 Software is “customer validation ready,”
allowing you to easily tailor the standard application to meet
your needs. Internal teams audit the Applied Biosystems
Quality Program regularly and independent auditors from
UL and PDA/ARC certify and document Applied Biosystems
processes for customers in regulated industries.* An audit is on
file in the PDA/ARC Repository.
Figure 2.
New, streamlined navigation makes SQL*LIMS v5 software easier to learn and use.
SQL*LIMS Software is the LIMS of choice for more than 30,000
professionals in 1,000 laboratories across
a wide range of industries
All informatics systems from Applied Biosystems are backed
by our global support, professional services, and informatics
specialists worldwide.
Figure 1.
SQL*LIMS v5 software includes expanded functionality for instrument calibration,
user qualification and reagent inventory tracking
*Underwriters Laboratories, Inc. (UL) is a leader in U.S. product safety
and certification worldwide. PDA is a non-profit international association
of scientists involved in developing, manufacturing, and regulating
pharmaceuticals/ biopharmaceuticals and related products. PDA developed
the standardised audit process used by suppliers of computer products and
services for regulated pharmaceutical operations. The Audit Repository
Center (ARC) serves as the global repository for all audits.
Contact
us
product news
Introducing
Tempus™ Blood RNA Tubes
Stabilise RNA profiles in whole blood for 5 days!
INFO
Tempus Blood RNA
Tubes enter: 125
T
he Tempus Blood RNA Collection Tube is
revolutionising gene expression measurement
by significantly extending your RNA profiling
capability. When combined with the ABI PRISM™ 6100
Nucleic Acid PrepStation, this unique solution
delivers a host of benefits:
» Stable RNA and transcript profiles in 3 mL of whole blood
for up to 5 days at room temperature
» Unequalled real-time PCR and microarray analysis results
» Fast, easy purification of total RNA from up to six Tempus
tubes simultaneously in 45 minutes
» Superior RNA quality
Or visit:
http://info.applied
biosystems.com/tempus
ABI PRISM 6100 Nucleic
Acid PrepStation
enter: 126
This instability is the result of metabolic activity that continues
in standard blood collection tubes post-draw (see figure 1).
Up-regulation of ~50-fold has been measured for some gene
targets after one hour at room temperature. Because blood is
often drawn at sites remote from the investigating team,
there is often a time lag (typically hours but sometimes days),
before the RNA isolation process can begin. During this time
lag, ex vivo changes in expression profiles can occur.
Measuring gene expression in whole blood is becoming an
increasingly important research tool. ‘Global’ measurement
of expression changes in whole blood by microarray
technologies permits the profiling of many thousands
of mRNA transcripts simultaneously in a single sample.
Real-time PCR assays based on TaqMan® probes or
SYBR® Green 1 Dye can be performed on a smaller number
of mRNA transcripts (also known as biomarkers), in a larger
population group to understand the importance of these
expression changes. This can be of significant help in gaining
an in-depth molecular profile of a disease, or understanding
the impact of various treatments.
For accurate measurement of gene expression profiles,
transcription must be stopped at the time of the blood draw.
The Tempus Blood Tube is designed for direct isolation of
3 mL of patient blood into a standard plastic, evacuated blood
collection tube containing the Applied Biosystems RNA
Stabilising Reagent. The RNA, and therefore the expression
or transcript profile, is stable in the collection tube for
up to five days at room temperature (18–25°C), or longer at
4°C (figure 2). The stabilised blood may also be frozen in
the collection tube for prolonged storage or transport.
Isolation of high-quality RNA from whole
blood samples is difficult
Recent work at Applied Biosystems1 has shown that the
task of isolating high-quality RNA is complicated further
because under ambient conditions, expression profiles are
unstable on a timescale of less than one hour.
Measuring gene expression in whole blood is becoming an increasingly important research tool. ‘Global’ measurement
of expression changes in whole blood by microarray technologies permits the profiling of
many thousands of mRNA transcripts simultaneously in a single sample.
Applied Biosystems has developed a unique RNA extraction
mechanism that generates the highest quality total
RNA and requires only a small number of steps
A simple 45-minute protocol on the ABI PRISM 6100 Nucleic Acid
PrepStation, purifies the RNA and generates high-quality nucleic
acid (A260/280 > 1.9) at yields of 2–8 µg/mL of human whole
blood. Up to six tubes can be processed simultaneously, producing
total RNA that is free of RT/PCR inhibitors and other enzymes.
The RNA is suitable for all downstream applications, including
real-time PCR and microarray analysis.
Safe, high-quality VACUETTE® tubes
The Tempus Blood RNA Tube was developed collaboratively
by Applied Biosystems and Greiner Bio-One GmbH, one of
the world’s leading suppliers of blood collection products well
known for both quality and safety. The tubes were the
first to be manufactured from the virtually unbreakable
polyethylene terephthalate (PET) plastic and can withstand
freeze/thaw procedures.
Figure 2.
Comparison of gene expression stability from blood stabilised in
Tempus Blood RNA Tubes and standard EDTA blood collection tubes.
The tubes were the first to be manufactured from the virtually
unbreakable polyethylene terephthalate (PET) plastic
and can withstand freeze/thaw procedures
RNA isolation Chemistry
Applied Biosystems has developed a unique RNA extraction
mechanism that generates the highest quality total RNA and
requires only a small number of steps.
Applied Biosystems RNA chemistry isolates a representative and
proportional selection of messenger, ribosomal and small nuclear
RNAs. It does not isolate tRNA, mammalian 5S ribosomal RNA,
highly degraded RNA, or any RNA species less than approximately
200 nucleotides. This, along with very low protein contamination
of the RNA, provides distinct advantages over other extraction
chemistries, in which tRNA and 5S ribosomal RNA often
comprise 15–20% of total RNA.
References
1. Tanner et al., Clin. Lab. Haem. 2002, 24, 337–341
To find out more about high performance,
low-cost prep solutions, visit:
Figure 1.
Gene expression profile in non-stabilised whole blood
(Tanner et al., Clin. Lab. Haem. April 2002, 24,337–341)
http://info.appliedbiosystems.com/prep01
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The GeneAmp® PCR System 2700 thermal cycler has been designed to meet
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Achieve
better, faster synthesis
and superior peptides
T
he ABI 433A Peptide Synthesis System, equipped
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Figure 1.
Improved synthesis of ‘difficult peptides’
*Terms and conditions for this special offer: Orders can be placed
by email, fax or phone quoting a purchase order number. Offer valid
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apply. All prices exclude delivery and local tax. E&OE.
For ordering, please use the part number and refer to the respective reference code below.
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4317033
HATU Kit
(19g HATU and DMF)
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GEN076521
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customer focus
German researcher
wins Applied Biosystems award
for excellence in mass spectrometry
In the eighties, he used plasma desorption mass spectrometry
for the analysis of intact biopolymers and, in more recent years,
has been working on Fourier transform ion cyclotron
resonance (FTICR) mass spectrometry. The results of this
work have given extraordinary insight into the biologically
active conformations of proteins and their pathophysiological
changes, for example, in the course of Alzheimer’s disease,
where it has recently provided the elucidation of a new
vaccine lead structure (Nature Medicine, 2002)”.
Above.
left to right:
Professor
Dr Juergen Grotemeyer,
University of Kiel;
Professor
Dr Jasna Peter-Katalinic,
University of Muenster;
Professor
Dr. Michael Przybylski,
University of Konstanz,
Dr Holm Sommer,
Applied Biosystems
G
erman researcher Professor Dr Michael Przybylski
is the latest scientist to receive a coveted award
sponsored by Applied Biosystems, which recognises
individuals who have made a significant contribution to the
development and application of mass spectrometry in life
sciences. Professor Przybylski, who is head of the Laboratory
of Analytical Chemistry at the University of Konstanz,
was selected by a scientific jury in recognition of his
outstanding work in the area of protein analysis.
The jury was appointed by the Board of the German
Society of Mass Spectrometry and led by Professor Dr. Jasna
Peter-Katalinic, who acknowledged his work in her award
speech: “In the past two decades, mass spectrometry has
undergone important developments and its use in genomics and
proteomics has effectively revolutionised life science research.
Novel concepts have allowed mass spectrometry to be used in a
more dynamic way and Professor Przybylski has contributed to
these developments from a very early stage.
More than 250 original research articles published
Professor Przybylski’s research is currently focusing on
proteomics of neurodegenerative proteins, applications of
mass spectrometry in immunology and the identification
of the chemical structures of molecular recognition by
antibodies. He has published more than 250 original research
articles, given approximately 100 invited lectures and is
co-editor of several scientific journals. His research is involved
in numerous international collaborations with European
laboratories and with the National Institutes of Health in the
US. He is adjunct guest professor of the Chinese and the
Hungarian Academy of Sciences, and has received an honorary
doctoral degree from the A.I. Cuza University of Iasi (Romania).
The award and a prize of 5,000 Euros was presented by
Professor Peter-Katalinic in a ceremony held at the UFZ
Centre for Environmental Research, Leipzig-Halle GmbH at
the Helmholtz Association, in Germany.
Applied Biosystems expresses its warmest congratulations to
Professor Przybylski and wishes him every success in the future.
“In the past two decades, mass spectrometry has undergone
important developments and its use in genomics and proteomics
has effectively revolutionised life science research”
customer focus
BioQualification
SM
Service
Supporting the Qualification process in your laboratory
with the appropriate documentation and agreements
ocumentation of proper installation of laboratory
equipment, and its intended use, is a basic
requirement in a regulated environment.
Good Laboratory Practices (GLP), Good
Manufacturing Practices (GMP), Good Clinical Practices (GCP),
ISO 9000 and other international standards define the
needs for an operation which at the end delivers the traceable
records and documents during the lifecycle of an instrument.
D
The Operational Qualification (OQ) or Instrument Performance
Verification (IPV) will follow immediately after the IQ. Now the
verification of the offered specifications, according to the
specification sheet of the instrument, proves that the instrument is
working as expected. Those steps are either outlined in detail directly
in the protocol or there is a reference taken to other supporting
documents, which are provided in the IQ/OQ package. Collateral
records and reports are added to the OQ protocol where applicable.
The ordered instrument has to be appropriate for the application
it is needed for. This is part of the Design Qualification (DQ).
The specification sheet is a valuable source of information about
the guaranteed characteristics of the system. During the sales
process a thorough review of the needed features compared
with the offered features, together with the Sales representative,
will lead into the purchase of the right system.
The installation done by a Field Service Engineer (FSE) includes the
set-up of the instrument, the verification of the installation
specification as outlined by Applied Biosystems and also the
introduction into the basic operation of the instrument. Those who
have fully attended the training session during the installation are
recorded in the Logbook as trained users.
At the point of installation an Installation Qualification (IQ)
is required. The IQ assists in documenting what had been
previously ordered and also been delivered. Is it the right model?
What about the purchased accessories? Is the software complete?
At what firmware is the system running? Which serial numbers
need to be recorded? After this has been noted down into the
protocol, the instrument will be installed according to
installation requirements.
The IQ/OQ protocols are always product specific and as the IQ/OQ
is very much tight into the on-site processes defined within the
laboratories SOP’s, the documentation is provided to our customers
as soon as the purchase is done. This gives the time to allow the
review of the protocol and to adopt the necessary changes to the
SOP’s in order to link all required documents smoothly into each
other. A pre-approval of the document by the quality personnel at
the customers’ site and the person executing the steps in the protocol
will document that this has happened.
An upfront delivered pre-installation guide in combination
with the requirements discussed during the sales process will
allow planning of the laboratory environmental operating
conditions. To have those conditions right is a prerequisite for
a successful installation. A check-up of the instruments basic
functions is the end of the IQ part.
As adequate maintenance of the system is demanded by the FDA,
other services are offered by Applied Biosystems such as Service
Agreements including Planned Maintenance (PM). To combine for
instance an annual PM with an annual OQ of the instrument is a
liked combination by those customers who have incorporated our
recommendation into their SOP’s.
Documentation of proper installation of laboratory
equipment, and its intended use, is a basic
requirement in a regulated environment
A subject that is often discussed between the quality department at
the customers’ site and the manufacturer is the question whether the
OQ has to be performed after a repair. To help in this discussion we
have worked out a list of spare parts for each instrument, which are
crucial for the instruments specifications.
Part of our quality philosophy is to always have
the best educated and knowledgeable
engineers employed
This spare parts list is available in the protocol for the customers
to judge to what extent an OQ has to be re-done in the event
of a repair, where one of those spare parts has been exchanged.
For all instruments installed by a FSE, the execution of the
IQ/OQ is part of the BioQualification package price. The FSE
is fully trained on the instrument and his/her training
certificates are available on request. Part of our quality
philosophy is to have always best educated and knowledgeable
engineers employed, this also requires training on the IQ/OQ
execution and a constant update on all changes happening to
the system.
The tools to align and adjust the instrument under PM
or service are carried by all FSE’s. Those tools are manufactured
by Applied Biosystems or available as standard tools on the
market. When needed both sorts of tools are calibrated
according to their calibration cycle. All tools that require
calibration are tracked in our system and are traceable to
international standards.
BioQualification products are available for a broad range
of instruments we offer (e.g. Real-Time PCR Systems,
Mass Spectrometers, Genetic Analysis Products and others),
and can be combined with the various Service Agreements
such as BioAssurance Plus, BioMaintenance , etc.
SM
SM
INFO
For more information:
Contact your local Applied Biosystems office,
Sales Representative or Field Service Engineer
Acknowledgement
Robert Keidl, Applied Biosystems, Germany
customer focus
High throughput
Genotyping
using ABI PRISM® 7900HT
Sequence Detection System
A partnership between a
CNRS laboratory and the
Applied Biosystems and
Genoscreen companies
ABI PRISM 7900HT Sequence Detection System
A
pplied Biosystems, Genoscreen (Lille-France) and the
Genetics of Multifactorial Diseases Laboratory
(Lille-France) initiated a collaborative project in
September 2003 using high-throughput genotyping to discover
new susceptibility genes for type 2 diabetes and obesity.
This project will include approximately 70 genes and up to
350 SNPs. The combined size of cohorts will be up to 2,000
individuals for each study. Thus, this project will require
completion of 700,000 genotypes.
Type 2 diabetes and obesity candidate genes were
selected based on several criteria including:
» chromosomal localisation near an obesity/type 2 diabetes–linked
locus in human or animal models
» expression profile in response to modification of the environment
in a variety of tissues (including adipocytes, hypothalamic
neurons, pancreatic beta cells or skeletal muscle)
» role in physiological pathways such as food intake, insulinresistance and insulin secretion
» targeted gene disruption or transgenic over-expression modifying
phenotypes associated with obesity and/or type 2 diabetes in
animal models.
This project will include approximately 70 genes and up to 350 SNPs. The combined size of
cohorts will be up to 2,000 individuals for each study. Thus, this project
will require completion of 700,000 genotypes
The “common disease, common variant” hypothesis for
multifactorial diseases (Reich and Lander 2001), implies that
typing of the most frequent SNP representative of each candidate
gene haplotype block (covering up to 20-30 kb or more) should
allow enough statistical power to detect linkage disequilibrium
(LD) between a functional SNP located in this block and the
complex trait under study. The association can be detected even if
the genotyped SNP is not itself functional but rather is in LD
with the causative variant(s).
The most informative, high frequency SNPs, regularly spaced
throughout the entire gene sequence from the 5‘ region to the
3‘ region of each candidate gene, were selected using the
SNPbrowser™ data visualisation tool. The selected SNPs are
genotyped in the case and control DNA samples and the genotype
and haplotype SNP frequencies are compared in subjects with
familial T2D versus non-diabetic subjects and also between obese
versus non-obese subjects. In addition, for each SNP, analysis of
variance of intermediate quantitative traits tests will be performed.
More than 100 obesity and type 2 diabetes related phenotypes are
available including BMI, leptin, insulin secretion and insulin
sensitivity indexes, lipids, insulin and glucose during oral glucose
tolerance test (OGTT).
In addition to bringing considerable amounts of novel genetic
information to the field of metabolic disease research, this study
demonstrates the viability and the efficiency of the ABI PRISM®
7900HT Sequence Detection System (TaqMan® assay technology
from Applied Biosystems) for high throughput genotyping
(up to 30,000 genotypes a day) and the technical advantages that
genomic platforms like Genoscreen can provide to researchers.
Figure 2.
The association can be detected even if the genotyped
SNP is not itself functional but rather is in
LD with the causative variant(s)
The combination of SNP selection strategy (SNPbrowser
software), SNP genotyping technology, a high throughput
SNP genotyping platform (Genoscreen), biostatistic resources
(Laboratory of multifactorial diseases) described here can
contribute to a powerful and rapid identification of potential
susceptibility genes for multifactorial diseases. Furthermore,
this approach can save time and manual labour.
Additional genetic and functional studies will be required to
identify the ‘disease causing’ SNPs that are associated with the
development of T2DM and obesity.
INFO
ABI PRISM 7900HT Sequence
Detection System enter 127
To download SNPbrowser Software visit:
www.appliedbiosystems.eu.com/mk/get/
aboptinsnpbrowser
Figure 1.
Identification of susceptibility genes for Obesity and type 2 Diabetes.
Related article can be found on:
Page 38
customer focus
Genomics
Spotlight
Dr Raija L.P. Lindberg, Head of the Clinical
Neuroimmunology Laboratory, The Department of
Research and Neurology, University Hospital of Basel
T
he main topics of work at the Clinical Neuroimmunology
laboratory are analysis and research on multiple sclerosis
(MS). The aim of our work is to comprehensively define
altered physiological pathways at various stages of development
of multiple sclerosis (e.g. during the development of lesions,
during relapses and remissions) in various tissue compartments.
We approach this by studying the gene expression patterns in
brain tissue and peripheral blood from MS patients, and in
brain and lymph node tissues in two animal models of
MS (delayed-type hypersensitivity (DTH) and experimental
autoimmune encephalomyelitis (EAE).
Our research is currently following two lines of enquiry.
The first aim is to get better insight into the pathogenic molecular
mechanisms of MS by comparing the gene expression profiles of
MS tissues to that of normal, non-neurological patients.
Defining altered pathways in the disease will ultimately lead to new
treatment strategies of MS. Secondly, we are looking for predictive
and diagnostic markers for the disease itself and its course.
Our aim is also to be able to estimate the response of various
treatments and, in that way, to optimise patient management and
hopefully prevent long-term damage in the central nervous system.
Ideally, these markers should be expressed in easily accessible
specimens, e.g. blood or urine, rather than for instance in
cerebrospinal fluid (CSF). Part of our research programme is linked
to clinical trials for candidate drugs and treatment regimens.
What populations are you using for your studies?
This laboratory is closely connected to the MS clinic in Basel
University Hospital. My colleagues are mostly clinicians who are
treating MS patients daily and consequently we have good access to
patient samples to use in our research. We are also studying autopsy
samples from MS patients and comparing them with samples
from other neurological diseases and control subjects.
We aim to verify the findings from the human studies with the
corresponding animal models of MS and test possible new treatment
strategies first with animal models before starting clinical trials.
“Our aim is also to be able to estimate the response of various treatments and,
in that way, to optimise patient management and hopefully prevent
long-term damage in the central nervous system”
Above: Dr. Raija L.P. Lindberg
“The aim of our work is to comprehensively
define altered physiological pathways at
various stages of development of multiple
sclerosis in various tissue compartments.”
Dr. Lindberg gained a PhD in Biochemistry at the
University of Turku, Finland, in 1986. From 1987
to 1990, she was Visiting Research Associate in the
Laboratory of Reproductive and Developmental
Toxicology, NIEHS, NIH, North Carolina, USA,
where she specialised in molecular biology. From 1990
to 1998, as an independent Research Associate in
Department of Pharmacology, the University of Basel,
Switzerland, she developed and studied a mouse model
for the human disease, Acute Intermittent Porphyria.
From 1998 to date she has been Head at the Clinical
Neuroimmunology Laboratory at Department of
Research and Neurology in Basel.
And this is where TaqMan® Gene Expression Assays
from Applied Biosystems come in?
Yes. I saw an advertisement shortly after the products became
available and realised that it was exactly what I needed at this stage
of the project. Technically, the dynamic measuring range of
microarrays is limited and may fail to detect subtle changes for
genes with very high or very low expression levels. Therefore we
used quantitative real-time PCR (RT-PCR) and the TaqMan Gene
Expression Assays to further verify leads from the array data.
What did you do before the TaqMan Gene Expression
Assays became available?
I either had to design the assays myself or run a pre-developed assay
reagents (PDAR) system if it was available. The problem is that
designing assays from scratch is so time-consuming and with the
time saved by the TaqMan Gene Expression Assays, I can get on
with the data analysis itself. In the early stages, not all the targets
we needed were available but most of the important ones were
and while I worked on these, the others were being developed.
More assays have become available and now all I need to do is to
browse the list for product numbers of genes of interest and then
just order. Unfortunately, at the moment all the assays are for the
well-defined sequences. For ESTs (expressed sequence tags) and
other unknown sequences I still need to design assays myself.
“More assays have become available and now all I need to do
is to browse the list for product numbers of genes
of interest and then just order”
Technologies used:
» ABI PRISM® 7000 Sequence Detection System
» TaqMan Universal PCR Master Mix
®
» TaqMan® Gene Expression Assays
What are your preliminary findings?
MS is characterised by the formation of disseminated areas of
demyelination and neuronal damage, called lesions or plaques,
therefore it has been defined as a focal disease. We have studied the
expression profiles not only in lesions but also in so-called normal
appearing white matter (NAWM), which is located in a vicinity
of lesions but looks macroscopically normal. Our main findings
from these studies provide molecular evidence of a continuum of
dysfunctional homeostasis and inflammatory changes in lesions and
NAWM, and support the concept of MS as generalised as opposed
to a focally restricted disease of the CNS. Moreover, our findings
corroborate that even in the progressive phase of disease, reparative
systems are active to compensate for structural damage.
What do these studies involve?
We are using a microarray technique to get comprehensive
information of the gene expression profiles altered in MS.
In parallel, we are using real-time PCR analysis to validate the
microarray findings and further evaluate the expression of
individual target genes.
What direction will your research now take?
At the moment we are using human assays as much as possible and
these are readily available as TaqMan Gene Expression Assays.
However, we will soon need to confirm that mouse and/or rat
models show the same phenomenon as we have seen in humans.
New possible treatment strategies need to be first tested in animal
models before proceeding to clinical trials. For that we will need
rodent alternatives to the TaqMan Gene Expression Assays.
Assays for other species are now becoming available, therefore we
will probably combine TaqMan Gene Expression Assays and
custom TaqMan Gene Expression Assays.
INFO
ABI PRISM® 7000 Sequence Detection
System enter 128
TaqMan® Universal PCR Master Mix enter 129
TaqMan® Gene Expression Assays enter 130
bio-highlights
Research using the
8500 Affinity Chip Analyzer
Presented at two
recent conferences
I
n January 2004, Applied Biosystems formally introduced
the 8500 Affinity Chip Analyzer for antibody
characterisation and protein interaction applications.
This instrument uses SpotMatrix SPR technology to enable
label-free measurement in real-time for kinetic analysis of
biomolecular interactions.
The first 8500 system was sold to Genentech (San Francisco,
CA) in January, which they have used to investigate various
protein-protein and protein-peptide interactions. Their research
using the 8500 Affinity Chip Analyzer was presented at two
recent conferences:
IBC’s Advances in Protein Science:
APS 2004 World Summit
» April 19-21, 2004 at the Boston Park Plaza, Boston, MA
CHI’s Beyond Genome 2004:
Proteomics - Addressing Challenges in Proteomic Analysis
» June 23-24, 2004 at the Fairmont Hotel, San Francisco, CA
Applied Biosystems will also be presenting data generated from
the 8500 Affinity Chip Analyzer at conferences worldwide.
Please contact your Applied Biosystems representative for
more information on this technology.
bio-highlights
Applied Biosystems
asks...
what are your
Real-time PCR needs?
B
etween 15 December 2003 and February of this
year we carried out a market research survey to
discover more about our customers' Real-time PCR
needs. First of all, we would like to take this opportunity to
thank all our customers who returned their completed
questionnaires. The valuable information that we received
was very much appreciated and will help us better match your
future Real-time PCR needs!
All the participants of the survey, who completed and returned
their questionnaire, had the chance to enter our prize draw to win
one of five Casio Exilim Z4 digital cameras.
The draw took place in February, and the five lucky winners were
presented with their digital cameras in March. We would like to
congratulate the following lucky customers and hope they have
fun with their new cameras:
AB-ONLINE >>>
News on demand
for integrated science
W
elcome to e-News, our on-line news channel.
Providing an exciting mix of features and links,
e-News is a valuable source of information for any
researcher working in the life science arena.
Each issue of e-News provides you with the latest innovations in
your area of interest, together with applications focused news to
help move your research forward.
» Application Focus: Provides the latest news on application
techniques and market focus
» Product Focus: Updates on our latest product releases
» Special Offers: Details of our current special offers
» Events: Updates on our forthcoming events in Europe
» Customer Relations: Features life science news plus links
to more information, support and training
To register for Small Molecules e-News visit:
http://news.appliedbiosystems.eu.com/LCMS_eNews
To register for Proteomics and Cell Biology e-News visit:
http://news.appliedbiosystems.eu.com/Pro_CB_eNews
To register for Assays e-News visit:
http://news.appliedbiosystems.eu.com/assay_enews
Dr Susana Benlloch, Hospital General, Alicante, Spain
To register for Forensics e-News visit:
http://news.appliedbiosystems.eu.com/forensic_enews
Mr Jérome Vicenzi, Cypher Science/TraceTag Europe, Paris, France
Dr Alessandra Movilia, Ospedale Civile di Legnano, Milan, Italy
Mr. Ludo A.B. Oostendorp, Streeklaboratorium
voor de Volksgezondheid voor Groningen en Drenthe,
Groningen, The Netherlands
To register for Genomics e-News visit:
http://news.appliedbiosystems.eu.com/genomics_enews
Dr. Olga Slamborová, KlinLab s.r.o, Prague, Czech Republic
bio-highlights
Narrowing in
on your mutations
INFO
visit:
http://myScience.
VariantSEQr
Resequencing System
enter 131
T
he breadth of genomic information now available
means a huge amount of the researcher’s time is
spent in gathering and organising this information.
In mutation detection particularly, literature and database
searches are necessary before concentrating on the intended
research project.
After collecting background information on the genes
or the pathway of interest, setting up the experimental
design requires diving into the literature and performing
validation experiments.
Applied Biosystems now offers two new tools, VariantSEQr™
Resequencing System1 and myScienceSM, to significantly ease
the work and to shorten the time between hypothesis
and result.
myScience
A free-of-charge online life science research environment for
genomic-based experiments is saving your time by providing
all necessary information in one place2. With a single query,
it allows you to gather critical information about a gene or
pathway with a direct link to a broad offering of information
connected to this gene:
» Finding relevant information on NCBI* and other online
information e.g. LocusLink genes, RefSeqs and NCBI
mRNAs for Human or equivalents for Mouse and Rat,
plus additional content such as Celera’s protein
classification information for these genes and articles
from key researchers
*National Center for Biotechnology Information
» Easy-to-use graphical map viewer allows to visualise
information from a single gene up to a whole chromosome
VariantSEQr Resequencing System
A complete application solution for detecting mutations or
variants in thousands of disease-related human genes.
» Accomplishing all resequencing projects with superior
data quality
» No primer design and validation processes required
» Identifying all variants using the most accurate
technology available
» Ready-to-use primer sets formulated at the working
concentration and analysis software that is fully integrated
with Applied Biosystems reagents and instruments
» Reducing the complexity of data analysis with relevant
information such as the reference sequence for all
amplicons provided with the analysis software
Acknowledgements
Beate Rätz,
Heinz Doersam,
Applied Biosystems,
Germany
Confidence values are assigned by a primer design algorithm
and take problematic areas into account (low complexity
repeat, GC content, GC count in localised regions, propensity
to form triple helices etc.). Each RSA (Resequencing
Amplicon) is designed to perform according to the assigned
confidence value when using recommended protocols and
Applied Biosystems equipment and reagents.
Applied Biosystems now offers two new tools to
significantly ease the work and to shorten the
time between hypothesis and result
» Applied Biosystems’ genomic products, such as
VariantSEQr Resequencing System can easily be
identified and selected based on the provided information
What information related to VariantSEQr is
available in myScience, and how do you find it?
Searching
There are four different ways to search for specific
VariantSEQr Resequencing Systems:
Search can be done by Keyword, Batch ID code numbers
(e.g. RefSeq and GenBank accession numbers), Chromosome
Location or by Molecular Function
Result list (RSS ID)
The Search Results (figure 1) are displayed as basic
information about the Resequencing Primer Set (RSS),
like gene identification information, type, size, number of
amplicons and percentage of coverage. More detailed
information about the gene is just one click away.
Detail Report Gene
From the Detail Report Gene page, a hyperlink “Gene
Name/Gene Symbol /ID” provides general information about
the gene, which would normally be collected by in depth
literature search, for example:
» Involvement in processes and gene function
» Background information about location and category
of the specified gene
» Hyperlinks to NCBI reports
Figure 1.
The assay results page contains detailed information about the Resequencing Set
Discover Your Gene Research Tools
on myScience today.
You are just a click away.
http://myScience.appliedbiosystems.com
A graphical view of the chromosomal region can be found using
the ‘map view’ hyperlink on that page.
Figure 2.
Detail Report resequencing primer set
Detail Report Resequencing Primer Set (figure 2)
Information about the resequencing primer itself set can be
accessed via hyperlink ‘RSS ID’.
Information about the amplicons in the set includes coordinates,
covered region, and a confidence value.
The information on the RefSeq public gene assembly and the
Celera Discovery System gene assembly of a specific gene is
mapped to a supertranscript called Target Region, including the
base sequence of that region. This information can be viewed in a
graphical display (figure 3).
With the ‘Export Results’ link the myScience research results data
can be saved into a text file to be exported to the researcher’s
computer, for archiving or further studies.
How to order VariantSEQr Resequencing Sets?
If you are registered and logged in at our online store
(www.appliedbiosystems.com/catalog) you can directly order the
selected VariantSEQr Resequencing Sets from myScience.
Figure 3.
Target Regions and RSA (resequencing amplicons) are depicted
in separate tiers with the Gene or RefSeq transcript as a reference
» Simply check the box in front of a Resequencing Primer Set
and click add to selected Shopping Basket
» Prices will reflect all your special conditions
» You will receive an immediate order confirmation
» You can track your shipments and order history online
Check out for the current VariantSEQr promotion at myScience
and get a 20% discount together with other attractive offers.
References
1. Mutation Analysis made easy, Biosystems Solutions Issue 8, 2003
2. Walk along the Chromosomes, Biosystems Solutions Issue 9, 2004
bio-highlights
LCMS Food and Environmental
Applications Binder
A
pplied Biosystems Mass Spectrometry systems are helping
scientists solve analytical problems with Mass Spectrometry
in clinical trial, food/beverage, and environmental
applications and bring unparalleled experience and product quality to
the broad field of testing, identification and quantification. To support
this we have produced a comprehensive Application Binder focusing
on Food and Environmental testing:
» The analysis of polycyclic aromatic hydrocarbons (PAHs) by
LC/MS/MS using an atmospheric pressure photoionisation source
» Detection of acrylamide in starch-enriched food with
HPLC/MS/MS
» Simultaneous analysis of multiple anti-bacterial drugs in food
products using LC/MS/MS
» Simultaneous determination of residues of approximately 100
pesticides and metabolites in fruit and vegetables by LC/MS/MS
» Determination of organophosphorous and organonitrogen
pesticides in fruit and vegetables by LC/MS/MS
» HPLC/MS/MS analysis of bitter acids in hops and beer
» Isolation and characterisation of anti-viral nucleoside compounds
from the Chinese herb taraxacum mongolicum
» Determination of N-nitrosamines in baby bottle rubber teats by
liquid chromatography-atmospheric pressure chemical ionisation
mass spectrometry
» Fast multi-residue pesticide analysis in soil and vegetable samples
» The combination of monolithic columns and modern
HPLC/MS/MS instrumentation for high chromatographic
throughput and sensitive detection of beta-agonists
» Determination of polar organophosphorus pesticides in aqueous
samples by direct injection using HPLC/MS/MS
» Detection of nitrofurane metabolites in food with HPLC/MS/MS
» Simultaneously characterising and quantifying chloramphenicol
and its metabolite using LC/MS/MS
» High sensitivity quantitation of metabolites of nitrofuran
antibiotics in animal tissue using LC/MS/MS
To download any of the application notes listed above visit
http://www.appliedbiosystems.eu.com/mk/get/ABSMPDFREG
bio-highlights
Applied Biosystems
Customer Training Courses
Call Jonathan MacBeath 01925 282530
email: [email protected]
Course
July
ABI PRISM 7000 Sequence Detection System
ABI PRISM® 7700 Sequence Detection System
Sept
01-02
ABI PRISM 7900 Sequence Detection System
12-13
®
Applied Biosystems 7300 & 7500 Real-Time PCR Systems
August
05-06
®
29-30
19-20
ABI PRISM 310 Genetic Analyzer
26-27
ABI PRISM® 3100 Genetic Analyzer
02-03
®
Oct
21-22
Reply no. 132
07-08
Reply no. 133
11-12
Reply no. 134
30-01 Oct
08-09
Reply no. 135
28-29
Reply no. 136
05-06
Reply no. 137
16-17
Applied Biosystems 3730 DNA Analyzer
01-03
SeqScape /GeneMapper Software
™
04 & 18
®
HID Course
Reply no. 138
10
07
Reply no. 139
15-17
13-15
Reply no. 140
Call Birgit Berenz +49 (0)6151 9670 5251
email: [email protected]
Course
July
August
Sept
Oct
API 2000 /API 3000 Systems*
08-09
05-06
23-24
14-15
Reply no. 141
API 4000™ System
06-07
03-04
21-22
12-13
Reply no. 142
Q TRAP System - Small Molecules
26-28
16-18
13-15
18-20
Reply no. 143
QSTAR® System - Small Molecules
07-09
4000 Q TRAP System
28-30
™
™
®
™
Q TRAP® System - Proteomics
BioCAD® Workstation/Vision™ Workstation
18-20
15-17
10-12
QSTAR® XL System - Proteomics
Voyager-DE™ PRO/STR Workstation
15-17
03-05
27-29
14-16
Dates on Application
Reply no. 144
20-22
Reply no. 145
12-14
Reply no. 146
26-28
Reply no. 147
Reply no. 148
Reply no. 149
* = 2 days training in-house + WebEx follow-up afterwards
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iScience. To better understand the
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our customers, Applied Biosystems
provides the innovative products, services,
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Biosolutions 9D - Applied Biosystems

Transcription

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