III Simposio Mexicano de Espectrometría de Masas 1

Transcription

III Simposio Mexicano de Espectrometría de Masas
1
III Simposio Mexicano de
Espectrometría de Masas
Proteómica Celular y Molecular
Ciudad de San Luis Potosí
8 al 12 de Noviembre 2009
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3
4
5
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iner.salud.gob.mx
ipicyt.edu.mx
cinvestav.mx
ccg.unam.mx
inmegen.gob.mx
ibt.unam.mx
insp.mx
biofound.org
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Programa y Resúmenes
III Simposio Mexicano
Mexicano de
Espectrometría de Masas
Proteómica Celular y Molecular
Ciudad de San Luis Potosí
8 al 12 de Noviembre 2009
Sociedad Mexicana de Proteómica AC
San Luis Potosí, S.L.P., México
www.smp.org.mx
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INFORMACIÓN GENERAL
Registro
Se llevará a cabo en el Salón Duque del Hotel Westin
Conferencias
Las conferencias Magistrales y Orales se llevarán a cabo en el Salón
Real San Luis “A” y los stands y posters en el Salón Real San Luis “B”.
Se anexa mapa del Hotel
Comidas
Las comidas (desayuno, comida) se incluyen a los participantes que
se hospeden en el Hotel Westin.
El comité organizador ofrecerá un Coctel de bienvenida en el Salón
Real San Luis y Patio central el día 8 de Noviembre después de la
Conferencia Inaugural.
También ofrecerá una Cena de Clausura el 12 de Octubre a las 20:00
horas en el Hotel Westin
Servicios de Café
Durante los días del Simposio, habrá café y refrescos disponibles
durante los recesos programados.
Entrega de Constancias
Las constancias para los expositores (ponentes) serán entregadas por
el coordinador de cada sesión. Las constancias para los asistentes se
entregarán el jueves 12 de octubre
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ÍNDICE
Pág.
Mensaje de la Mesa Directiva
2
Comité Organizador
3
Programa del Simposio
4
Resúmenes de Conferencias Plenarias
11
Resúmenes de Sesiones Orales
47
Resúmenes de Presentaciones en Cartel
62
Índice de autores
110
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Mensaje de la Mesa Directiva
Estimados participantes,
La presente mesa directiva de la Sociedad Mexicana de Proteómica desea darles
la más cordial bienvenida al 3er Simposio de Espectrometría de Masas,
Proteómica Molecular y Celular. En conjunto hemos preparado un programa
donde participarán destacados científicos internacionales y nacionales con el fin
de escuchar diferentes temas de interés en nuestro campo de estudio. Es
indudable que los avances de la tecnología proteómica han sido espectaculares y
sus aplicaciones constituyen una promesa para estudiar el proteoma de humanos,
microorganismos, plantas y animales. Sus aplicaciones a la medicina, entre otras
muchas funciones, son por ahora una excelente alternativa en: a) la identificación
de nuevos biomarcadores, b) descubrir mecanismos moleculares involucrados en
la patogenia de enfermedades aun poco entendidas, y c) en el descubrimiento de
nuevos fármacos. Por todo esto, deseamos hacer de este Congreso un encuentro
placentero basado en un programa científico de muy buena calidad y amplias
oportunidades de interactuar entre grupos de trabajo de la academia e iniciativa
privada en México y otros países. En esta ocasión, además, hemos dedicado un
espacio especial a los trabajos de los estudiantes e investigadores que se inician
en el área de la proteómica, conscientes de que ellos son el futuro de esta
disciplina en nuestro país. Finalmente, deseamos mostrar nuestro agradecimiento
a las compañías que con su apoyo han contribuido al éxito de este Congreso.
Atentamente
Mesa Directiva (2008-2010).
Sociedad Mexicana de Proteómica
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SOCIEDAD MEXICANA DE PROTÉOMICA A.C.
Presidente: Luis M. Terán, INER
Vice-Presidente: Humberto Lanz Mendoza, INSP
Tesorero: Gerardo Corzo, IBT-UNAM
Secretaria: Ana Paulina Barba, IPICyT
Vocales:
José Luis Gallegos Pérez, Inmegen
Bronwyn Jane Barkla, IBT
Luis González de la Vara, Cinvestav-Irapuato
Roberto Arreguín Espinosa, Instituto de Química, UNAM
COMITÉ ORGANIZADOR LOCAL
Ana Paulina Barba de la Rosa, IPICyT
Fabiola León Galván, IPICYT
Alicia Chagolla, CINVESTAV-Irapuato
Ing. Gabriela Lizbeth Melendez Govea, IPICYT
L.C.C. María Teresa Gallegos Cepeda, IPICyT
L.D.G. Sofía González Cabrera, IPICYT
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PROGRAMA
Registro
Domingo 8 y lunes 9 de noviembre: Salón Duque a partir de las 9:00 h
Domingo 8 de Noviembre
8:30 - 9:50
Registro
Curso Pre-Simposio, Salón Real San Luis “A”
9:00 – 10:30
Programa Educacional I
Principios básicos de cromatografía y su acoplamiento a
Espectrometría de masas.
Francisco Rojo, Facultad de Química, UNAM
10:30 – 11:30
Conceptos básicos en espectrometría de masas, CL
multidimensional y otras metodologías analíticas utilizadas en
el análisis proteómico
José Luis Gallegos Pérez, INMEGEN
11:30 – 12:00
Receso
12:00 – 13:00
Programa Educacional II
Métodos de análisis de proteínas por espectrometría de
masas (PMF y métodos MSMS)
Alicia Chagolla, CINVESTAV-Irapuato
13:00 – 14:00
Programa Educacional III
Bioinformática
Ernesto Pérez Rueda, IBT-UNAM
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13:30 – 15:30
LUNCH
16:50 – 17:00
Inauguración
17:00 – 18:00
Conferencia Inaugural
Presentador: Dr. Luis M. Terán
Dr. Hans Peter Mock
Evaluation of plant genetic resources using proteomic
approaches
18:00 – 20:00
Inauguración de la Sección de Posters
Coctel de Bienvenida
Lunes 9 de noviembre
9:00 – 9:50
Lectura Plenaria II
Dr. Richard Caprioli
Molecular Profiling/Imaging of compounds in tissues by
mass spectrometry: Assessing spatial and temporal
changes.
9:50 – 10:40
Lectura Plenaria III
Dr. Cesar Batista
Proteomics analysis of amphibian skin secretion using
high resolution mass spectrometry.
10:40 – 11:00
Receso
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11:00 – 12:15
Simposio 1: Proteómica Clínica
Presentador: Dr. Luis M. Terán
1. Dr. Richard Caprioli
Biomarker discovery using MALDI mass spectrometry: A
study of human melanoma
2. Dr. Genaro Pimienta
Proteomic identification of novel biomarkers for HER2positive breast cancer
3. Dr. Genaro Pimienta
Proteomic investigation of inactive and active JNK2
12:15 – 13:30
Simposio 2: Proteómica Biomédica
Presentador: Dr. Humberto Lanz Mendoza
1. Dr. Juan Pablo Reyes Grajeda
Proteomic analysis of oxidative stress in a diet-induced
hypercholesterolemic model
2. Dra. Rosa M. del Angel
Proteómica del Dengue
3. Dr. Sergio Encarnación
Proteomics for gynecological cancer study
13:30 – 15:30
Comida
15:30 – 16:50
Sesiones Orales I
17:00 – 19:00
Sesión de Posters
19:00
Cena
6
Martes 10 de noviembre
9:00 – 9:50
Lectura Plenaria IV
Dr. Jack Throck Watson
Interleafing chemistry and mass spectrometry to
characterize the structure of folding intermediates of
cysteinyl proteins
9:50 – 10:40
Lectura Plenaria V
Dr. Sixue Chen
Proteomics of plant redox regulatory networks
10:40 – 11:00
Receso
11:00 – 12:15
Simposio 3: Proteómica de enfermedades infecciosas
Presentador: Dr. Gerardo Corzo
1. Dra. Victoria Pando
Proteomics technology for virus research
2. Dr. Manuel Rodríguez
Functional characterization of ubiquitylated protein
complexes using Tandem ubiquitin binding entities
(TUBEs): Toward the ubiquitin interactome
3. Dra. Cecilia Silva Sánchez
Flujo de trabajo para el descubrimiento de biomarcadores en
proteómica
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12:15 – 13:30
Simposio 4: Proteómica de Plantas
Presentador: Dra. Bronwyn Barkla
1. Dra. Bronwyn Barkla
Quantitative proteomics of tonoplast from the halophyte
Mesembryanthemum
crystallinum
reveals
novel
associations and functions of glycolytic enzymes in plant
salt tolerance
2. Dra. Silvia Valdés
Análisis proteómico de la respuesta al estrés hídrico en
células de Bouteloua gracilis
3. Dr. Luis González de la Vara
Separation of calcium-dependent protein kinases from
beetroot plasma membranes according to their
hydropathy, and their identification by mass
spectrometry
13:30 – 15:30
Comida
15:30 – 16:50
Sesiones Orales I
17:00 – 19:00
Sesión de Posters
19:00
Cena
Miércoles 11 de noviembre
9:00 – 9:50
Lectura Plenaria VI
Dr. Hans Peter Mock
Characterization of plant co-chaperones with homology to
the human protein HOP (heat shock-organizing protein)
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9:50 – 10:40
Lectura Plenaria VII
Dra. Maria C. Prieto Conaway
Advantages of high mass accuracy, resolving power and
dynamic range of the MALDI LTQ Orbitrap for tissue
imaging experiments.
10:40 – 11:00
Receso
11:00 – 12:10
Simposio 5
Presentador: Dr. Luis González de la Vara
1. Dr. Hans Vissers
A data independent approach towards the qualitative
and quantitative profiling of complex protein mixtures
2. María C. Prieto Conaway
A novel pressure ion trap for high throughput protein
identification and highly sensitive analysis of drugs and
metabolites
12:10 - 13:30
Sesión de Negocios
13:30 – 15:30
Comida
16:50 – 19:00
VISITA GUIADA AL CENTRO DE SAN LUIS
19:00
Cena
Jueves 12 de noviembre
9:00 – 9:50
Lectura Plenaria VIII
Dr. Luis M. Terán
Proteómica de secreciones nasales de niños con
influenza A
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9:50 – 10:40
Lectura Plenaria IX
Dr. Robert Ventzki
3D-Gel electrophoresis, a new approach to protein
analysis
10:40 – 11:00
Receso
11:00 – 12:15
Simposio 6: Proteómica de microorganismos
Presentador: Dr. Sergio Encarnación
1. Dra. Yolanda López Vidal
Proteínas únicas y comunes entre la micobacterias no
tuberculosas de origen clínico y ambiental.
2. Dr. Sergio Encarnación
Proteome, phosphoproteome,
Rhiobium etli.
and
secretome
in
3. Dra. Clara Inés Espitia Pinzón
Interacciones patógeno-hospedero en tuberculosis. Un
abordaje proteómico.
12:15 – 13:30
Simposio 7: Nuevas tecnologías
Presentador: Dr. José Luis Gallegos Pérez
1. Dr. Michael L. Easterling
Top-Down Proteomics, a multiple-platform primer
2. Dr. Jack Throck Watson
Recent Developments in MS/MS Technology
13:30 – 15:30
Comida
15:30 – 16:50
Sesiones Orales
17:00 – 18:00
Clausura
18:00 – 19:00
Cena Clausura
10
RESÚMENES
CONFERENCIAS PLENARIAS
Salón Real San Luis “A”
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L01
EVALUATION OF PLANT GENETIC RESOURCES USING PROTEOMIC
APPROACHES
KATJA W ITZEL1, STEPHANIE KASPAR1, DIANA W EIER1, W INNIE W ESCHKE1, UDO
SEIFFERT2, ANDREA MATROS1 & HANS-PETER MOCK1
1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben,
Germany. 2 Fraunhofer-Institute IFF, Magdeburg, Germany
The major focus of the IPK Gatersleben is the collection, preservation and
evaluation of plant genetic resources with an emphasis on cereals. The wide
genetic diversity represented in plant gene-banks is central for further breeding
efforts towards a larger number of traits, such as enhanced stress tolerance or
improved nutritional quality of crop plants. Within recent years, methods for the
comprehensive analysis of transcripts, proteins and metabolites of cereals have
been introduced and are constantly improved to gain novel information within
fundamental or applied research. The presentation will focus on two examples for
the application of proteomics performed on barley. In a first approach, the genetic
diversity of barley for contrasting salt tolerance was evaluated using mapping
populations. Differences in the proteome were followed in contrasting parental lines
and selected off-springs to identify potential candidates related to salt tolerance.
Furthermore, the heterologous yeast system was used to identify barley gene
products complementing a salinity sensitive mutant strain. Candidate genes are
currently verified in transgenic approaches in barley and by mutant studies using
Arabidopsis. In a second study changes in the proteome of developing barley
grains were followed using a label-free LC-MS approach, which yielded a highly
complex multidimensional data-set. For the elucidation of statistically significant
and objective kinetic patterns during grain developmental processes multivariate
statistics was applied.
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L02
MOLECULAR PROFILING/IMAGING OF COMPOUNDS IN TISSUES BY MASS
SPECTROMETRY: ASSESSING SPATIAL AND TEMPORAL PROCESSES
Richard Caprioli
Vanderbilt University School of Medicine, Nashville, TN USA
The spatial and temporal aspects of molecular processes in cells and
tissues play an enormous part in the biology that defines living systems. Profiling
and Imaging MALDI MS provides an effective means to measure and assess those
dimensions on a molecular basis, including peptides, proteins, lipids, metabolite as
well as others. The technology is extraordinarily high throughput with high
molecular specificity and is an excellent discovery tool. It provides the capability of
mapping the location of specific molecules directly from fresh frozen and formalin
fixed tissue sections. For example, utilization of this technology provides spatial
information across a tissue section for a target protein expression or for a signature
of multiple proteins and can be used to correlate changes in expression levels with
specific disease states or drug response. Molecular patterns can be directly
correlated to known histological regions within the tissue, a technique termed
histology directed molecular profiling.
In the case of frozen tissues specimens, cryostat sections (~10 µm thick)
are thaw-mounted on flat metallic target plates, and matrix automatically deposited.
This can be done in a histology-directed manner to bring into play the expertise
and experience of pathologists to obtain molecular profiles from discrete areas of
tissue. Formalin fixed tissues can also be analyzed by first exposing them to an
antigen retrieval step. In the imaging mode, high density laser ablation of an
ordered array of spots over the entire tissue gives rise to a 2-dimensional ion
density map (or image) with 30-80 µm lateral resolution in which location and
relative abundance of a given analyte is displayed. From the analysis of a single
section, images at virtually any molecular weight may be obtained.
This presentation will discuss several applications of this technology,
including examples of discovery in mouse developmental models and the profiling
of human tumors, characterizing protein differences between tumor grades, and for
the creation of 3-D protein images. In addition to MALDI ToF MS and MS/MS
instrumentation, the capabilities of ion mobility MS and FTICR MS for profiling and
imaging of tissues will be discussed. The technology has been applied to drug
targeting and metabolic studies and the measurement of concomitant protein
changes in specific tissues after systemic drug administration. Finally, we explore
the correlation of lipid and protein changes in several systems in both health and
disease.
13
L03
PROTEOMICS ANALYSIS OF AMPHIBIAN SKIN SECRETIONS USING HIGH
RESOLUTION MASS SPECTROMETRY
Cesar V. F. Batista
Unidad de Proteómica, Instituto de Biotecnología, Universidad Nacional Autónoma
de México, Cuernavaca, México. E. mail: [email protected]
Over 20 years have past since peptides with antimicrobial activity were first
identified in skin secretions of the African clawed frog Xenopus laevis. Since that
time, several hundred such antimicrobial peptides have been isolated from the skin
of anurans (frogs and toads) belonging to several families ranging from the
primitive Leiopelmatidae (tailed frogs) to the highly derived Ranidae (true frogs).
However, the number of species investigated and the number of bioactive
compounds are still small. Mexico is one of the 18-mega diverse countries of the
world. With over 200,000 different species, Mexico is home of 10–12% of the
world's biodiversity. Mexico ranks first in biodiversity in reptiles with 707 known
species, second in mammals with 438 species, and fourth in amphibians with 290
endemic species. Nevertheless, almost 400 different amphibian species can be
found in this country. Only in Morelos state 38 species were classified in different
families, which is estimated to contain over four thousand unknown bioactive
components in the skin secretion of these amphibians. Frog skin secretion has
been found to be a rich source of agents with antimicrobial (bacteria, fungi,
parasite and virus), anticancer, insulin releaser and smooth muscle contractor
properties, among others. The total secretion contains approximately one hundred
components constituting a very complex molecular mixture that require efficient
methods and technologies for large-scale analysis. Mass spectrometry has
become a powerful tool to analyze complex mixture of peptides and proteins during
the last decade, basically due to developing of high resolution and versatile mass
analyzers. In this work we present a large-scale analysis of the skin secretions
from Mexican amphibians using a high resolution Orbitrap-FT mass spectrometer
to determine the number of components and the chemical structure of novel
bioactive peptides. Hundreds of components were partially characterized and
many full sequences were obtained from the species Hyla eximia, Pachymedusa
dacnicolor, Lithobates spectabilis, Lithobates forreri and Lithobates spustulosus.
Antibacterial activities were also accessed using resistant bacteria from clinical
isolates. Three peptides isolated from L. spectabilis showed a potent activity
against multi-resistant Pseudomonas aeruginosa to all commercially available
antibiotics in Mexico.
Acknowledge: This work was partially financed by grants from DGAPA IN-221508
to CVFB.
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L04
PROTEOMIC INVESTIGATION OF INACTIVE AND ACTIVE JNK2
1,2
Pimienta G, Ficarro SB, Gutierrez GJ, Bhoumik A, Peters EC, Ronai Z,
2
Pascual J.
1
Current Address: Department of Molecular Biophysics and Biochemistry, Yale
University New Haven, CT. USA
2
Previous Address: Burnham Institute for Medical Research, La Jolla, CA. USA
The c-Jun N-terminal kinases (JNKs) are ubiquitous proteins that phosphorylate
their substrates, such as transcription factors, in response to physical stress,
cytokines or UV radiation. This leads to changes in gene expression, ensuing
either cell cycle progression or apoptosis. Active phospho JNK1 is the main in vivo
kinase component of the JNK cascade, whereas JNK2 is presumed not to
participate as a kinase during JNK signalling. However, there is evidence that JNK
isoforms interact functionally in vivo. Also, a recent chemical genetics investigation
has confirmed that JNK transient activation leads to cellular proliferation, whereas
a sustained one is pro-apoptotic. Here we investigate the phosphorylation pattern
of JNK2, with protein biochemistry tools and tandem mass spectrometry. We
choose to focus on JNK2 because of its reported constitutive activity in glioma
cells. Our results indicate that purified JNK2 from transfected nonstressed 293T
cells is a mixture of the mono-sites pThr183 and pTyr185 of its activation loop and
of pThr386 along its unique C-terminal region. Upon UV stimulation, its
phosphorylation stoichiometry is upregulated on the activation loop, generating a
mixture of mono-pTyr185 and the expected dual-pThr183/pTyr185 species, with
the pThr386 specie present but unaltered respect to the basal conditions.
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L05
BIOMARKER DISCOVERY USING MALDI MASS SPECTROMETRY: A STUDY
OF HUMAN MELANOMA
Richard Caprioli and William Hardesty
Vanderbilt University School of Medicine
The aim of this work is to determine a specific protein signature from stage
III melanoma tumors that distinguish and classify tumor and control lymph node
and that correlate to aggressive tumors through patient survival data. Patients that
present with stage III disease, metastasis to the regional lymph nodes, are at a
much higher risk for recurrence and disease progression. Currently, these patients
exhibit a range in survival and treatment response. Diagnostic and prognostic
clinical approaches to distinguish this stage of disease for are predominantly
macroscopic and molecular classification has focused mainly focused on genetic
mutations.
In this study, proteomic data from 69 stage III melanoma and 17 disease
free lymph nodes acquired by histology-directed MALDI IMS is used to classify
tumor from control lymph node and to molecularly sub-classify stage III disease by
tumor aggressiveness. The histology directed MALDI technology allows a
pathologist to examine a serial, histological stained tissue section and guide and
confidently target the cellular regions of interest. MALDI matrix is then applied
directly onto the tissue at the regions selected by the pathologist. Results of
multiple classification models between control lymph node and stage III melanoma
show a return of an average recognition capability of 97% and a cross-validation
result of 92.5%. Using a Cox-Proportional Hazard model, 9 proteins were found
significantly (p<0.05) associated with survival and 3 proteins associated with
disease recurrence. This set was combined to generate a multiplex molecular
signature to group patients into poor (lower 50%) and favorable (upper 50%)
survival and recurrence sets.
16
L06
PROTEOMIC IDENTIFICATION OF NOVEL BIOMARKERS FOR HER2- POSITIVE
BREAST CANCER
ф1
2
3
3
1
Pimienta Genaro , Yien Gu , Xu Sun , Jianjun Hu , Kim Min-Sik , Chaerkady
1
4
4
5
Raghothama , Gucek Marjan , Cole Robert N , Sukumar Saraswati , Pandey
1,6
Akhilesh
2¶
and Chen Hexin
1
McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry,
Johns Hopkins School of Medicine, Baltimore, Maryland, USA
2
Department of Biology, University of South Carolina, Columbia, South Carolina, USA
3
Department of Computer Science and Engineering, University of South Carolina,
Columbia, South Carolina, USA
4
The Johns Hopkins School of Medicine, Mass Spectrometry and Proteomics Facility,
Baltimore, Maryland, USA
5
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine,
Baltimore, Maryland, USA
6
Departments of Pathology and Oncology, Johns Hopkins School of Medicine, Baltimore,
Maryland, USA
ф
Current address:
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven,
Connecticut, USA
The receptor tyrosine kinase HER2 is an oncogene commonly amplified in breast
cancer and in many cases indicative of poor prognosis in patients. Accordingly, its
over-expression in mammary epithelial cell lines is determinant of a tumorigenic
phenotype. Various quantitative proteomic studies have been reported that explore
the role of HER2 in conferring a cancerous proteomic phenotype. Most of these
investigations have entailed comparing differences in protein profile by combining
two-dimensional gel electrophoresis and tandem mass spectrometry. In this study
we have used SILAC-based proteomics to determine the proteomic profile of
HER2-expressing mammary epithelial cells isolated from a transgenic mouse.
From a short-list of 23 proteins with a relevant annotated function and a distinctive
SILAC ratio, we highlight that the up-regulation of Plastin 2, Thymosin beta 4 and
Tumor protein D52 supports the notion of a tumorigenic phenotype that we observe
by cell characterization. In addition, we show by in-silico microarray data analysis
that the down-regulation of Gelsolin 1 and the up-regulation of Retinol-binding
protein 1 can be used to predict the probability of metastasis-free survival in breast
cancer patients. These two proteins may thus be considered novel cancer
biomarkers.
17
L07
PROTEOMIC ANALYSIS OF OXIDATIVE STRESS IN A DIET-INDUCED
HYPERCHOLESTEROLEMIC MODEL
J. P. Reyes-Grajeda1, K. Calderon-Gonzalez1, J. L. Gallegos-Perez1, A.
Jiménez-Corona2, A. Moreno2, S. Damián-Zamacona3, & Jaime Mas-Oliva3
1. Unidad de Proteómica Médica. Instituto Nacional de Medicina Genómica,
INMEGEN, 2. Instituto de Química, Universidad Nacional Autónoma de México
(UNAM), 3. Instituto de Fisiología Celular, Universidad Nacional Autónoma de
México (UNAM). E-mail: [email protected]
In Mexico the general prevalence of hypercholesterolemia in the open population is
in the order of 26.5 % with direct evidence of its involvement in the process of
atherosclerosis.
This disease presenting a multifactorial etiology involves the interaction between
genetic and environmental factors that in turn modulate the various functions of
different cell types and inflammatory molecules within the vessel wall. Since the
specific participation of the different cell types in the formation of the
atherosclerotic plaque, directly correlated to the presence of both normal and
abnormal proteins is still under active investigation, the need for efficient detection
systems in plasma are becoming critical.
Here, we report a proteomic analysis of differentially expressed proteins in the
plasma of normal and hypercholesterolemic rabbits (New Zealand), fed either with
a control or a high-cholesterol diet for a period of sixteen weeks.
The proteomic analysis of plasma proteins carried out with both groups of
experimental animals shows the fact that several proteins reach statistical
significance for differential expression based on a DIGE gel analyses (using a
threshold level of +two-fold and -two-fold) obtained from both samples after being
fed with the high-cholesterol diet.
The MS data analysis from this last group clearly shows an increase in a series of
proteins associated with the presence of oxidative stress, process that has been
associated with the development of atherosclerosis.
Therefore, the principal aim of this line of investigation has been the search of
homologous proteins related to human disease such as the ones involved with
abnormalities in lipid metabolism and cardiovascular calcification, both processes
directly related to the development and progression of atherosclerosis.
18
L08
PROTEÓMICA DEL DENGUE
Jorge Reyes-del Valle, Martha Yocupicio-Monroy, Sofía Alcaraz-Flores,
Raúl Agis-Juárez, Salvador Chávez-Salinas, Rosa M. del Angel
La fiebre del dengue es la infección viral transmitida por mosquitos más ampliamente
distribuida en el mundo. Se estima que anualmente esta enfermedad afecta a 100
millones de personas, siendo 2.500 millones las que viven en áreas de riesgo de
transmisión de la enfermedad. La infección por dengue puede manifestarse en tres formas
clínicas de gravedad creciente, la fiebre clásica del dengue, que se caracteriza por fiebre
alta, cefalea, artralgia, mialgia, linfadenopatía y leucopenia; el dengue hemorrágico que
se distingue de la fiebre clásica por la fuga plasmática y la trombocitopenia simultanea y el
síndrome de choque por dengue que se manifiesta por hemo-concentración y en los
casos más graves, por fallo circulatorio, estado de choque (síndrome de choque por
dengue) y la muerte en cierta proporción de casos. El agente causal de la fiebre del
dengue y sus complicaciones es el virus del dengue (DEN), miembro de la familia
Flaviviridae y del género flavivirus. Se reconocen cuatro serotipos del virus (DEN-1, DEN2, DEN-3 y DEN-4) y dentro de cada serotipo varios genotipos. Morfológicamente, DEN es
una partícula esférica de aproximadamente 50 nm de diámetro, la cual contiene una
cápside de 30 nm rodeada por una envoltura lipídica. La superficie del virus contiene dos
proteínas, la proteína E (de la envoltura) y la proteína M (de membrana). La glicoproteína
E contiene la mayoría de los determinantes antigénicos del virus y es indispensable en la
entrada viral. El genoma de DEN consiste en una cadena de RNA de polaridad positiva de
aproximadamente 11kb, con una estructura Cap tipo I, m7GpppAmpN2,, en su extremo 5’,
y carece de la cola de poli (A) en su extremo 3’. En lugar de poli (A), DEN tiene una
estructura de tallo y burbuja muy estable y rica en nucleótidos CU. En el extremo 5’ del
genoma viral, la región no traducida (RNT) está constituida por aproximadamente 100
bases (de 89 nt para DEN-2 y de 101 nt para DEN-4), mientras que en el extremo 3’ la
RNT comprende aproximadamente 400 nucleótidos (384 para DEN-4 a 466 para DEN-1).
Como para todos los virus de cadena positiva, el RNA genómico de DEN es infectivo. La
traducción del genoma viral da origen a una poliproteína, que es procesada co- y posttraduccionalmente, para dar origen a 10 proteínas virales maduras (C, prM y E, y las
proteínas no estructurales NS1, NS2A, NS2B, NS3, NS4A, NS4B y NS5). Dado que
DEN, como muchos otros virus, codifica para un número limitado de proteínas tiene que
utilizar diversas proteínas y organelos celulares para entrar, traducirse, replicarse y formar
nuevas partículas virales. Estas moléculas celulares determinan en gran medida que el
virus sea infectivo, el tropismo, la susceptibilidad y la virulencia. Con la finalidad de
identificar algunas de las proteínas celulares que participan en la entrada de DEN
aislamos proteínas con afinidad a la proteína de la envoltura E mediante cromatografía de
afinidad. El análisis por MALDI-ToF de las proteínas de membranas aisladas nos permitió
identificar a las proteínas de choque térmico HSP90 y HSP70. Por otro lado, separamos
las proteínas con afinidad a la RNT3’ de DEN-4 con la idea de conocer algunas de las
proteínas celulares que participan en la replicación viral. El análisis por MALDI-ToF nos
permitió identificar a las proteínas: la proteína de unión al tracto de polipirimidinas, la
proteína disulfuro isomerasa y a calreticulina. La función de estas proteínas en el ciclo
replicativo viral ha sido confirmada mediante su silenciamiento y/o su sobreexpresión.
19
L09
PROTEOMICS FOR GYNECOLOGICAL CANCER STUDY
Sergio Encarnación, Juan Carlos Higareda, Alberto Checa Rojas, Magdalena
Hernández y Alberto Carlos Ramírez Torres
Laboratorio de proteomica. Centro de Ciencias Genómicas. Cuernavaca, México
Email: [email protected]
During the carcinogenic process, several genetic and epigenetic changes occur in
the cell, all of which derive in altered expression, post-translational modification, or
activity of the proteins, causing an aberrant cellular behavior that leads to cancer.
Proteomics opens new venues in the research for diagnostic, disease progress,
and treatment response in cancer. Currently, we are using proteomic tools in the
study of gynecological cancers in an attempt to identify proteins that change their
expression or post-translational modifications when compared against healthy
controls. These can be used as specific biomarkers of these pathologies.
We have taken three different approached to this problem. We searched for subtle
changes in the general protein expression pattern, analyzed and identified the
secreted proteins of cellular lines of cervical cancer “in vitro” and “in vivo”; and we
analyzed auto-antibodies of cervical cancer patients in search of possible
biomarkers.
PROTEOME
We first examined differential protein expression patterns of six cellular lines using
two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted
laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. Protein
expression was evaluated using PDQuest 2-D software. The common expressed
protein spots were identified with MALDI-TOF mass spectrometer and the peptide
mass spectra identification was performed using the Mascot program searching the
Swiss-prot or NCBInr databases. A total of 74 proteins related to the neoplasic
phenotype were detected by this method, including Annexin A2, Protein disulfideisomerase, Vinculin, Ezrin, 14-3-3 protein zeta/delta, Heat shock 70 kDa protein 1
and 78 kDa glucose-regulated protein.
20
SECRETOME
To establish the secretome we used 2D SDS-PAGE, MALDI-TOF mass
spectrometry and high-resolution liquid chromatography coupled to Electrospray
Ionization Tandem Mass Spectrometry (HPLC/ESI-MS/MS) to analyze and identify
the proteome contained in the secretions of three cell lines. For this, “in
vitro”(mono-layer culture), and “ex vivo”, meaning nu/nu mice xeno-transplants,
were generated. A total of 50 secreted proteins were identified, including CaMkinase II, RuvB-like 2, Annexin A5, T-complex protein, Peroxiredoxin-6 and
Vasohibin-2.
POSSIBLES BIOMARKERS
To search biomarkers of cervical cancer, we performed western-blots of 2D SDSPAGE from cell lines using auto-antibodies obtained from cervical cancer patients.
Here, proteins which presented an immunogenic reaction were identified with
MALDI-TOF mass spectrometry and HPLC/ESI-MS/MS, and evaluated like
possible biomarkers. A total of 6 candidate proteins were identified, Heat shock
protein 75 kDa, T-complex protein 1 subunit zeta, Succinate dehydrogenase
[ubiquinone] flavoprotein subunit, Heterogeneous nuclear ribonucleoprotein H and
Annexin A2.
Part of this work was supported by CONACyT grant 60641 and DGPA-PAPiiT
grant IN222707.
21
L10
INTERLEAFING CHEMISTRY AND MASS SPECTROMETRY TO
CHRACTERIZE THE STRUCTURE OF FOLDING INTERMEDIATES OF
CYSTEINYL PROTEINS
Jack Throck Watson
Emeritus Professor, Departments of Biochemistry and of Chemistry
former Director, MSU Mass Spectrometry Facilities, Michigan State University
East Lansing, MI 48824, e-mail: [email protected]
Some types of structure elucidation require chemical modification of the analyte so
that subsequent analysis by mass spectrometry can provide relevant data. In this
talk, well-established chemical reduction procedures and cyanylation chemistry will
be described as a means to degrade cystinyl proteins in a controlled manner so
that mass spectrometry of the resulting degradation products can be used to
establish the connectivity of cysteines in various cystines (disulfide bonds) in the
original protein molecule. In another example, cyanylation chemistry will be
described as a means to capture various cysteinyl intermediates during the
oxidative folding of BPTI. The degree and locus of various regions of local
structure (alpha helix/beta sheet) can be recognized by ‘spray-painting’ the analyte
with D2O, ‘freezing’ the positions of incorporated deuterium atoms during controlled
degradation of the labeled analyte, and subsequent analysis of the degradation
products by mass spectrometry. An example of this last procedure will be
illustrated by pulse-labeling of Long Arg3 Insulin-like Growth Factor (LR3IGF-I) with
D2O, minimizing back-exchange of deuterium atoms by imposing low pH (pD) and
low temperature, degrading the labeled LR3IGF-I with pepsin, and analyzing the
peptic fragments by mass spectrometry.
Keywords: Mass spectrometry, cyanylation, disulfide bonds, cystinyl proteins.
22
L11
PROTEOMICS OF PLANT REDOX REGULATORY NETWORKS
Sixue Chen1*, Mengmeng Zhu1, Johanna M. Strul2, Sophie Alvarez1, Justin
Goldsmith1
1
Department of Biology, University of Florida, Gainesville, FL 32610, USA
2
Life for Science Program, University of Florida, Gainesville, FL 32610, USA
* e-mail: [email protected]
Protein redox regulation is increasingly recognized as an important switch of
protein activity in yeast, bacteria, mammals and plants. In this study, we have
identified proteins with potential thiol switches involved in plant hormone signaling,
which is essential for plant growth, development and defense. Methyl jasmonate
(MeJA) treatments led to enhanced production of hydrogen peroxide in Arabidopsis
leaves and roots, indicating in vivo oxidative stress. With monobromobimane
(mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total
of 35 protein spots that displayed significant redox and/or total protein expression
changes were isolated. Using LC-MS/MS, the proteins in the spots were identified
in both control and MeJA treated samples. By comparative analysis of mBBr and
SyproRuby gel images, we were able to differentiate proteins that were redox
responsive from proteins that displayed abundance changes in response to MeJA.
Interestingly, stress and defense proteins constitute a large group that responded
to MeJA treatment. In addition, many of the cysteine residues involved in the
disulfide dynamics were mapped based on the tandem MS data. Recently, we
have applied the technology to the study of guard cell redox proteins and their
functions in stomatal movement. Characterization of redox proteins and their
cysteine residues involved in the redox regulation allows for a deeper
understanding of plant hormone signaling networks.
This work was supported by University of Florida and a National Science
Foundation grant to S. Chen.
23
L12
PROTEOMICS TECHNOLOGY FOR VIRUS RESEARCH
Victoria Pando Robles, Victor Higareda, Odelia Benitez, Humberto Lánz, Javier
Izquierdo.
Centro de Investigación en Enfermedades Infecciosas. Instituto Nacional de Salud
Pública., Cuernavaca, México. Email: [email protected]
Advances in genomics and proteomics and the use of bioinformatics will enable
more detailed molecular studies in the pathogenesis, etiology and pathology of
viral diseases. All viruses as obligatory cellular parasites require host cell factors to
complete their replication cycles, and they also have to contend with the antiviral
defense mechanisms of the host. Overall changes in the host cellular proteome
upon virus infection result in a cascade of signaling events and pathway activation.
Cell type- and tissue-specific responses are also characteristic of infection and can
be classified based on the differential expression of genes and proteins between
normal and disease states. The identification of differentially expressed proteins
during infection is also critical in discovering potential biomarkers within infected
bodily fluids. Biomarkers can be used to monitor the progression of infection, track
the effectiveness of specific treatments and characterize the mechanisms of
disease pathogenesis. On the other hand, viruses are released from infected cells
in the form of virions, which contain all the essential factors necessary for initiating
infection in a new target cell. Recently, different studies report that some viruses
like influenza and HIV contain cellular proteins in the virion or post-translation
modification, although the functional significance of these packaged host proteins
has not yet been determined. Many factors play into the complexity of viral
pathogenesis. Here, we report the strategy to study cellular proteomes in response
to rotavirus infection and dengue infection, using standard and quantitative
approaches.
24
L13
FUNCTIONAL CHARACTERISATION OF UBIQUITYLATED PROTEIN
COMPLEXES USING TANDEM UBIQUITIN BINDING ENTITIES (TUBEs):
TOWARDS THE UBIQUITIN INTERACTOME
M.S. Rodriguez1, R. Hjerpe1, F. Lopitz-Otsoa 1, F. Aillet1, M. Torres-Ramos1, V.
Lang1, P. F. Elortza2, England3.
1
Proteomics Unit and 2Proteomics Platform CICbioGUNE-CIBERhed, Bizkaia,
Spain, 3Institut Pasteur, Plate-forme de Biophysique des Macromolécules et de
leurs Interactions and CNRS, URA2185, Paris, France. Email:
[email protected]
Post-translational modification with ubiquitin is one of the most important
mechanisms of regulation of protein stability and function. The high reversibility of
this modification is a major obstacle for the isolation and characterization of
ubiquitylated proteins. To address investigations on post-ubiquitylation
mechanisms, we have developed tandem-repeated ubiquitin binding domains
(TUBEs) based on ubiquitin-associated domains (UBA). TUBEs display a
remarkable increase in affinity for polyubiquitin chains, allowing an efficient
purification of ubiquitylated proteins from cell extracts, tissues and organs in native
conditions. Moreover, TUBEs protect ubiquitin-conjugated proteins (such as p53
and IκBα) from proteasomal degradation and de-ubiquitylating activity present in
cell extracts, increasing the purification efficiency. More importantly, TUBEs allow
us to follow stimuli dependent oscillations of ubiquitylation. These new 'molecular
traps' have been adapted to MS analysis to further characterize macromolecular
complexes regulated by ubiquitylation. With this purpose various cell lines were
exposed to genotoxic insults and accumulated polyubiquitylated proteins were
efficiently isolated and analyzed using this approach. Our results validate this new
technology for the study of ubiquitin-regulated processes.
25
L14
FLUJO DE TRABAJO PARA DESCUBRIMIENTO DE BIOMARCADORES
EN PROTEÓMICA
Silva Sánchez C.
Laboratorios Centrales, UGPM. CINVESTAV-IPN.
Av. IPN 2508, Col.San Pedro Zacatenco. C.P. 07360, México, D.F.
email: [email protected]
Las proteómica es una herramienta promisoria para la identificación de nuevos
biomarcadores que pueden mejorar el diagnóstico y el seguimiento de una
enfermedad a través de sus diferentes etapas. En el flujo de trabajo para su
descubrimiento, verificación y validación, la espectrometría de masas juega un
papel muy importante. En la etapa de descubrimiento, el objetivo es obtener el
mayor número de proteínas candidato a biomarcador, por lo cual los
espectrómetros de masas a emplearse deben ser muy sensibles y precisos.
En laetapa de verificación, se debe confirmar que los biomarcadores obtenidos
son verdaderos indicadores del estadío biológico de la muestra, por lo que los
equipos de espectrometría de masas deben ser capaces de identificar
solamente los biomarcadores obajetivo. El objetivo de esta presentación es
mostrar el flujo de trabajo desarrollado para las etapas de descubrimiento y
verificación de biomarcadores empleando un 4800 plus MALDI TOF-TOF y un
3200 QTRAP (AppliedBiosystems/MDS Analytical Technologies) en la Unidad
de Genómica Proteómica y Metabolómica del CINVESTAV-IPN.
26
L15
QUANTITATIVE PROTEOMICS OF TONOPLAST FROM THE HALOPHYTE
MESEMBRYANTHEMUM CRYSTALLINUM REVEALS NOVEL ASSOCIATIONS
AND FUNCTIONS OF GLYCOLYTIC ENZYMES IN PLANT SALT TOLERANCE
B.J. Barkla, R. Vera-Estrella, M. Hernández-Coronado, O. Pantoja
Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3,
Colonia Miraval, Cuernavaca, Morelos, México, 62250. Email:
[email protected]
To increase understanding of the role of the tonoplast in plant salt tolerance and with
the aim to identify proteins involved in the complex network of transporter regulation
to enhance vacuolar Na+ sequestration, we exploited a targeted quantitative
proteomics approach. Two dimensional Differential In Gel Electrophoresis (2DDIGE) analysis of Free Flow Zonal Electrophoresis separated tonoplast fractions
from control and salt-treated Mesembryanthemum crystallinum plants revealed the
surprising membrane association of glycolytic enzymes, aldolase and enolase,,
along with expected subunits of the V-ATPase. Western blot analysis confirmed coordinated salt-regulation of these proteins and chaotrope treatment indicated a
strong tonoplast association. Reciprocal co-immunoprecipitation studies revealed
that the glycolytic enzymes interacted with VHA-B of the V-ATPase and aldolase
was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP.
To investigate a physiological role for this association the Arabidopsis cytoplasmic
enolase mutant, los2, was characterized. Plants were salt-sensitive and showed a
specific reduction in enolase abundance in tonoplast from salt treated plants.
Moreover, tonoplast isolated from mutant plants showed an impaired ability for
aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic
proteins with the tonoplast may not only serve to channel ATP to the V-ATPase but
also directly upregulate H+-pump activity.
Acknowledgments
Mass spectra were obtained at the INMEGEN Medical Proteomics Facility with the
assistance of Dr. Jose Luis Gallegos Pérez. We thank Dr. Victoria Pando for
guidance with the DIGE method and Dr. Susana López for access to the Typhoon
Imager. This work was supported by CONACyT grants 49735 to B.J.B. and 57685
to R.V-E, and DGAPA IN221308.
27
L16
PROTEOMIC ANALYSIS OF THE RESPONSE TO OSMOTIC STRESS IN
BOUTELOUA GRACILIS
1
S.E. Valdés Rodríguez., 1 A. Guerrero Rangel., 2 A. Aguado Santacruz, 1 A
Chagolla López.
1
Departamento de Biotecnología y Bioquímica, Cinvestav, Km. 9.6 Libramiento
Norte, 36500, A.P. 629, Irapuato, Gto. México. 2Campo Experimental Bajío,
INIFAP – SARH, Apartado postal 112, Celaya, Gto. 38110, México.
[email protected]
Blue Grama (Bouteloua gracilis) is an important forage grass that grows in arid
and semiarid zones in México. The tolerance of Bouteloua gracilis for high-dry
environments makes it an ideal candidate for studying the molecular mechanisms
by which plants respond to water stresses. Recently, a chlorophyllic cell line
(‘TADH-XO’) from Bouteloua gracilis was developed (Aguado Santacruzz et al.,
2000). This represents an ideal system to analyze the cellular responses to water
deficit. In an attempt to study water stress responsive proteins in B. gracilis, we
compared the changes in the expression of proteins of cultured Bouteloua cells
adapted to grow in a medium supplemented with high molecular weight
polyethylene glycol (21% PEG), versus the modifications induced in cells abruptly
transferred to the same medium. PEG is a molecule commonly used to mimic
water stress because it reduces water availability without penetrating the cell. Cell
samples were harvested from stressed and control cultures for proteome analysis
by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Approximately,
forty proteins spots were significantly altered in each stress treatment, with respect
to the control cell culture. The proteins of differential expression were identified by
Mass Spectrometry. The MS/MS was performed on an ESI-QToF Mass
Spectrometer (Waters). Protein identification and functional categorization B.
gracilis proteomes and genomes have not been extensively characterized.
However, protein sequences entries for similar proteins expressed in plants could
be used, providing a reasonable amount of sequence homology by amino acid
sequences. The identified proteins were associated with a variety of functions,
including energy and metabolism, protein synthesis and degradation, cell defense,
cell growth/division, and transport. They showed important changes in expression
depending on the type of stress. ROS scavengers were up regulated in both types
of stress, suggesting that the antioxidative system play a role in protecting cells
from oxidative damage following exposure to osmotic stress. Chloroplast proteins
are over-represented in our study, suggesting that the chloroplast might be
28
involved in many of the cell´s responses to osmotic stress. Our chlorophyllic
system could help revealing the cellular mechanisms underlying the high drought
tolerance of B. gracilis.
Reference
Aguado-Santacruz GA., Cabrera-Ponce JL., Ramírez-Chávez E., León Ramírez
CG., Rascón-Cruz Q., Herrera-Estrella L., Olalde-Portugal V. 2001 Cell Report
20:131-13
This project was financial supported by CONACYT-Gobierno del Estado
Guanajuato (28555).
29
L17
SEPARATION OF BEETROOT PLASMA MEMBRANE PROTEINS ACCORDING
TO THEIR HYDROPATHY AND THEIR IDENTIFICATION BY MASS
SPECTROMETRY
L.E. González de la Vara1, B. Lino Alfaro1, A. Chagolla López2
1
Departamento de Biotecnología y Bioquímica, Cinvestav-Irapuato, Irapuato,
México, 2Cinvestav-Irapuato, Irapuato, México. Email: [email protected]
In order to identify proteins by MS, it is desirable to separate them previously. This
is mostly achieved by two-dimensional gel electrophoresis. In the case of
membrane proteins, this technique is not universally applicable, since the
detergents compatible with it are often unable to extract integral membrane
proteins with acceptable yields. We have then developed a method to separate
membrane proteins according to their hydropathy. It is based in the property of the
detergent Triton X-114 to efficiently extract membrane proteins at low temperature;
whereas at a temperature above 22° (“cloud point”) two phases are formed, in
which extracted membrane proteins partition themselves according to their
hydropathy. If this partition is performed serially, each protein is distributed in the
different fractions following the binomial distribution law (1). We have applied this
method to separate beetroot plasma membrane proteins, analyzing the proteins in
each fraction by SDS-PAGE. We found that the most abundant proteins (water
channels and H+-ATPases) are hydrophobic as anticipated. Hydrophilic proteins
are very diverse, in contrast to amphiphilic proteins which are few (1). Single
proteins from 1D-electrophoresis gels were immunodetected: they distributed
themselves in the different fractions following the binomial law, as expected. Their
hydropathies were then estimated from these distributions. Although some proteins
in these partition fractions have been identified by ESI-Q-ToF MS/MS after being
separated by 1D-electrophoresis, it is sometimes convenient to purify further these
proteins by ion-exchange chromatography and gradient centrifugation. Several
proteins, partially purified this way, have been identified by MS/MS, some Ca2+dependent protein kinases (which could phosphorylate and thereby regulate the
activity of the H+-transporting ATPase [2]) among them. (1) González de la Vara
LE, Lino B (2009) Anal Biochem 387: 280-86. (2) Lino B, et al. (1998) Planta
204:352-59.
30
L18
CHARACTERIZATION OF PLANT CO-CHPAERONES WITH HOMOLOGY TO
THE HUMAN HOP (HEAT SHOCK-ORGANIZING PROTEIN)
HANS-PETER MOCK
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben,
Germany. 2 Fraunhofer-Institute IFF, Magdeburg, Germany
.
31
L19
ADVANTAGES OF HIGH MASS ACCURACY, RESOLVING POWER AND
DYNAMIC RANGE OF THE MALDI LTQ ORBITRAP FOR TISSUE IMAGING
EXPERIMENTS
Maria C. Prieto Conaway
Thermo Fisher Scientific, San Jose, CA, USA. Email:
[email protected]
This presentation outlines the advantages of using high resolution mass
spectrometry (HRMS) with accurate mass for tissue imaging experiments. The
tissue imaging workflow is described in detail, from sample sectioning, MALDI
matrix application, data acquisition and data processing. Two different types of
studies will be discussed. One is a small molecule application, the use of tissue
imaging in the study of drug distribution and its metabolites in mice. The other is
the use of tissue imaging for the study of physiologically active peptides in
neuronal tissue of invertebrates.
Methods:
1) Sample preparation for the drug distribution study – Nude mice bearing
transplanted human head and neck FaDu tumors were treated with either
irinotecan, methylselenocysteine (MSC), both drugs in combination, or non-treated
(control). The drug cofactor MSC has been shown to increase irinotecan efficacy
against tumors. The tumors were excised and frozen sections (12 micron) were
thaw-mounted on to non-conductive glass slides. MALDI matrix was sprayed on
top of the tissue and full MS and MS/MS data were collected in the OrbitrapTM
analyzer. MS3 ion trap data was also acquired.
In a separate small molecule study, a whole rat section is analyzed, to highlight the
advantages of using MALDI imaging as compared with the alternate technique of
Whole Body Auto Radiography.
2) Sample preparation and workflow for the neuropeptide study involved rapid
dissection of neurosecretory tissue (i.c. insect neurohemal organ) in isotonic
sucrose solution, mounting the tissue on a glass slide, controlled spraying of the
air-dried tissue with concentrated MALDI matrix solution, loading specimen into the
MALDI source of an MSn system equipped with an Orbitrap analyzer, setting-up
32
imaging methods by determining targeted tissue areas of interest, spatial
resolution, molecular mass range and molecular mass resolution for the detection
in the FTMS (Orbitrap) detector, acquisition of mass spectra, analysis of data using
ImageQuestTM software to generate (single or composite) images of the distribution
of peptide(s) of interest and confirmation of identity of selected peptides by MS2
and/or MSn sequencing directly from imaged tissue sample.
Results:
The results illustrate that high mass accuracy and high mass resolving power of
the Orbitrap analyzer are achievable in analyses directly from tissue, such as in
imaging experiments. The MALDI LTQ Orbitrap mass spectrometer allows for both
peptide localization as well as peptide identification/sequencing directly from
tissue, with <3ppm mass accuracy. At resolving powers of 60,000 (defined @ m/z
400), isobaric peptides are separated and their individual masses mapped,
showing different localization according to function, all within a ~2 mm area of
insect brain. For small molecule applications MALDI imaging was able to detect
both parent drug and metabolites, show differences associated to different drug
treatments, in agreement with results from separate electrospray work and
previously published results. The analysis of whole body sections by imaging mass
spectrometry illustrates the advantage over Whole Body Auto Radiography,
because mass spectrometry differentiates between drug and metabolites, in
addition to showing localization to different organs.
Key words: mass spectrometry imaging, neuropeptides, Orbitrap detector, drug,
metabolite localization in tissue, anti-cancer drug.
33
L20
TOP-DOWN PROTEOMICS, A MULTI-PLATFORM PRIMER
Michael L. Easterling1
1
Bruker Daltonics, Inc. 40 Manning Road, Billerica,MA 01821
Although the idea of “top-down” processing intact proteins has been around for
some time, only recently have software and hardware advances allowed this thesis
to be pursued as a viable workflow. While there are many differing opinions about
the most efficient way to gather information from top-down protein work, the most
conventional misconception is that it requires the use of high performance
instrumentation. Indeed, almost any kind of mass spectrometer capable of
interfacing either MALDI or ESI can be used to generate data from intact proteins.
The challenge for the modern mass spectrometrist is to determine what kind of
“hammer” to apply to the problem at hand. “Lower end” analyzers such as ion
traps that offer lower resolving power and mass accuracy have been given new life
by recent advances in the combination of advanced ion fragmentation techniques
such as electron transfer dissociation (ETD) and increased analytical performance,
and are unquestionably capable of performing top-down quality control (QC) roles.
Time-of-flight instrumentation coupled with electrospray ionization (ESI) has
recently seen architectural improvements that allow resolving powers in excess of
50,000 allowing charge state and, ergo, mass determination of larger proteins even
under fast chromatographic conditions. MALDI-TOF has seen recent
methodological advances that allow protein terminal sequencing that rivals
standard Edmund sequencing techniques in terms of sequence coverage. Finally,
the MS arsenal is completed with Fourier Transform (FTMS) based instruments
which remain the most flexible and evaluative form of analyzer for the top-down
protein chemists. This discussion will focus on how each of these platforms play
an integral role in advancing top-down strategies and how each can be used in an
integrated whole protein strategy to compliment bottom-up workflows.
34
L21
A NOVEL DUAL PRESSURE ION TRAP FOR HIGH THROUGHPUT
PROTEIN IDENTIFICATION AND HIGHLY SENSITIVE ANALYSIS OF
DRUGS AND METABOLITES
Maria C. Prieto Conaway
Thermo Fisher Scientific, San Jose , CA, USA. Email:
[email protected]
A new source design coupled to a dual pressure ion trap features increased
sensitivity, with enhanced cycle time while maintaining excellent fragmentation
spectral quality. In proteomic studies this translates into substantial increase in
the detection and identification of both proteins and unique peptides. For small
molecule applications it can provide limits of detection down to 25 femtogram
on column and analysis of complex samples, such as tissue homogenates, on
fast chromatographic scales.
The dual pressure ion trap is described in this work and compared to existing
ion trap platforms for both types of applications.
To illustrate a proteomics study, a complex digest (C. elegans proteome) is
analyzed for protein identification (1). The small molecule application is a study
of irinotecan (Camptosar®, Pfizer) and its metabolites in liver tissues, drug
which has strong anti-tumor activity against a variety of human tumors.
Methods: Proteomics study -C. elegans homogenates were digested using a
previously described protocol. Proteolytic digests were separated by reverse
phase chromatography at 300 nL/min of 2-25% acetonitrile in 0.1% formic acid
gradient to a packed tip column. Gradient lengths of 60 and 180 min were
used. For data-dependent acquisition, the method was set to automatically
analyze the top 10 most intense ions observed in the MS scan.
Small molecule study - Nude mice were treated with irinotecan,
methylselenocysteine (MSC), both drugs in combination, or not treated at all.
The drug cofactor MSC has been shown to increase irinotecan efficacy against
tumors. Liver tissues were homogenized, protein precipitated and centrifuged
before separating by reverse phase chromatography. Compounds were eluted
using a 10-35% organic solvent gradient or 10-90% organic solvent gradient
over 5 or 15 min, respectively.
35
Results: The increased sensitivity and faster cycle times of the dual pressure
ion trap show not only increase in detection and identification of both proteins
and unique peptides, but also increased detection of low abundance peptides.
Faster cycle times on this new instrument allowed for higher throughput with
more proteins identified in the 60 min gradient.
In the study of irinotecan and its metabolites, a total of eight compounds were
analyzed using a fully automated data-dependent experiment which provided
excellent structural elucidation of both the parent drugs and their respective
metabolites. The ratio of irinotecan to its SN-38 metabolite in the livers of mice
treated only with irinotecan agrees with irinotecan to SN-38 ratios reported
previously for plasma and liver. Less irinotecan (parent drug) was extracted
from liver tissue in ‘drug plus MSC’ treated mice as compared to those from the
drug-only treatment. These findings suggest that MSC treatment resulted in a
reduction of irinotecan accumulation in the liver. Since MSC and related
Selenium-containing compounds are known to reduce the toxicity of certain
chemotherapeutic agents and to enhance the cure rate of tumors, these
findings are relevant to the protective mechanisms of MSC and are being
further investigated.
References:
1) Second, T., Blethrow, J., Schwartz, J., Merrihew, G., MacCoss, M., Swaney,
D., Russell, J., Coon, J., and Zabrouskov, V.; Dual-Pressure Linear Ion Trap
Mass Spectrometer Improving the Analysis of Complex Protein Mixtures;
Analytical Chemistry 2009.
36
L22
PROTEOMIC ANALYSIS OF PATIENTS SUFFERING INFLUENZA A
INFECTION
Luis M. Terán
Influenza viruses (IV) are major cause of respiratory infection in humans and result
in substantial illness, death and economic burden throughout the world. There are
two major types of human influenza viruses, A and B with influenza A strains
responsible for seasonal or pandemic influenza. Humanity first understood the
potential danger of influenza in 1918–1919, when a disease killed over 40 million
people around the globe.
Three other influenza pandemics have occurred on 1957, 1968, and now in early
2009. To date however, the mechanisms by which IV cause such a high morbidity
and mortality are not fully understood. This presentation will discuss the use of
proteomic technology in the analysis of nasal secretions derived from children
suffering naturally acquired seasonal influenza A infection. Findings reveal that the
composition of nasal secretions in influenza A virus respiratory infections is
different from that when children are healthy.
In addition, preliminary data on the analysis of biological samples obtained from
patients infected with the novel influenza A (H1N1)v virus will be presented.
37
L23
3D-GEL ELECTROPHORESIS, A NEW APPROACH TO PROTEIN ANALYSIS
Robert Ventzki1, Sjouke Hoving2, Jörg Bernhardt1,3, Jan van Oostrum4, and Josef
Stegemann1
1
Ernst-Moritz-Arndt University, Institute for Microbiology, 17487 Greifswald,
Germany
2
Novartis Institutes for BioMedical Research, Developmental & Molecular
Pathways, 4002 Basel, Switzerland
3
DECODON GmbH, 17489 Greifswald, Germany; 4Zeptosens AG, 4108
Witterswil, Switzerland. E-mail: [email protected]; www.3D-gel.com
Introduction
2-DE separation has not been considered suitable for large-scale comparative
protein expression studies due to its limited throughput. We have developed a
high-throughput 2-DE analysis method and instrument based on 3D geometry gel
electrophoresis. With the 3D-gel instrument in its last phase of development and
testing, we are investigating and initiating applications in mole-cular discovery,
clinical diagnosis, pharmacology and toxicology, like protein monitoring during
disease development and screening of drug candidates for their effect on protein
expression.
Methods
Following conventional isoelectric focusing (IEF), up to 36 IPG-strips are arrayed
on the top surface of a 3D-gel body and samples are transferred electrokinetically
to the gel. SDS-PAGE occurs in vertical direction perpendicular to the loading
surface. For optimum resolution and sensitivity, proteins are labeled according to
differential in-gel electrophoresis (DIGE) protocols and detected online using laserinduced fluorescence (LIF). As the proteins pass a laser-illuminated detection
plane in the 3D-gel, images are acquired by a digital camera and recorded as a 3D
image stack. Image processing software draws vertical sections out of the 3D
image stack, representing a series of 36 conventional 2-DE slab gels. Image
matching and statistical analysis is performed using DECODON Delta2D software.
38
Results
To our knowledge, the 3D-gel is the only method for high-throughput 2-DE analysis
with online LIF detection, providing results directly in electronic format without any
further gel processing steps. A specific thermal management ensures that SDSPAGE occurs under identical conditions for all samples, thereby permitting the
immediate comparison of the separation patterns without elaborate software
corrections. We tested the instrument for protein expression analysis of
Haemophilus influenzae treated with antibiotics. Image matching revealed an
unprecedented congruence of the separation patterns, leading to precise and
highly reproducible results. A unique way to apply the instrument is to introduce the
sample index (n = 1...36) as a third parameter to the analysis, i.e., denoting the
time, concentration, or dose of the sample treatment. The 3D-gel quantitatively
measures the protein abundance as a function of that parameter, making it a
valuable tool for large-scale comparative protein analysis.
Innovative aspects
• All samples of a series are analyzed simultane-ously in the same 3D-gel: Direct
comparability of separation patterns, no gel-to-gel variations, significant
improvement in the detection of small differences in protein expression.
• Online data collection by photodetection: Results are immediately accessible
without time-consuming gel incubation and documen-tation (no fixing, staining,
destaining, scanning).
• A single 3D-gel accommodates up to 36 IEF-prefractionated samples: High
throughput, easy handling, cost saving.
References
(1)
Robert Ventzki and Josef Stegemann,
High-throughput separation of DNA and proteins by 3-D geometry gel
electrophoresis, Electrophoresis, Dec. 2003, 24:4153-4160
(2)
R. Ventzki, S. Rüggeberg, S. Leicht, T. Franz, and J. Stegemann,
Comparative
2-DE
protein analysis in a 3-D geometry gel, BioTechniques, March 2007, 42:271-279
(3)
J.
Stegemann,
R.
Ventzki,
A.
Schrödel,
and
A. de Marco, Comparative analysis of protein aggregates by blue native
electrophoresis and subsequent SDS-PAGE in a 3-D geometry gel, Proteomics,
May 2005, 5:2002-2009
39
3D-gel electrophoresis workflow. Step 1: A series of 36 fluorescently labeled
protein samples is separated by conventional IEF. Step 2: All 36 IPG-strips are
arrayed in a slotted plastic grid and placed on the 3D-gel. Electrophoresis is
activated and samples are transferred to the 3D-gel. During SDS-PAGE, laserinduced fluorescence is detected online by a digital camera and recorded as a 3D
image stack. Step 3: Software generates vertical sections of the 3D image stack,
representing a series of 36 conventional 2-DE gels.
40
L24
IDENTIFICACIÓN Y ANÁLISIS COMPARATIVOS DE LOS PROTEOMAS DE
MICOBACTERIAS NO TUBERCULOSAS DE AISLAMIENTOS CLÍNICO Y
AMBIENTALES.
Dra. Yolanda López Vidal
Programa de Inmunología Molecular Microbiana, Facultad de Medicina,
Universidad Nacional Autónoma de México.
Las micobacterias no tuberculosas (MNT) son aquellas especies que no pertenecen al
complejo m. tuberculosis y que son distintas a M. leprae . las MNT tiene la capacidad de
causar patologías a nivel pulmonar, cuyos signos y síntomas son indistinguibles de la
tuberculosis. La prueba cutánea al derivado proteico purificado (que se basa en una
respuesta de hipersensibilidad de tipo retardado) es de poco valor en el diagnostico en
virtud de que un resultado positivo a esta prueba podría deberse a una respuesta inmune
inducida ya sea por MNT o por M. tuberculosis. Las proteínas de M. tuberculosis se han
estudiado para resolver las desventajas asociadas al uso del este reactivo. Sin embargo,
se sabe poco sobre la proteínas de MNT. El objetivo del presente fue identificar proteínas
únicas de cepas de MNT aisladas con mayor frecuencia de enfermedad pulmonar
humano y agua, así como las comunes con M. tuberculosis H37Rv mediante la
comparación de proteomas, de modo que fuera posible seleccionar proteínas con el
potencial de ser utilizadas como biomarcadores de infección.
Las cepas de micobacterias de aislados clínicos M. avium y M. abscessus de enfermedad
pulmonar y seis aisladas de agua para uso y consumo M. nonchromogenicum, tipo I, tipo
II, M. gordonae, M. peregrinum, M. avium aisladas de agua de la Ciudad de México y M.
nonchromogenicum ATCC 19550.
Las cepas de MNT se cultivaron hasta la fase logarítmica. El sobrenadante del cultivo y el
paquete celular se procesaron para extraer las proteínas , éstas se separaron por
electroforesis bidimensional (2D-PAGE) y los perfiles resultantes se analizaron in sílico
para localizar proteínas únicas y comunes con M. tuberculosis H37Rv. Algunas proteínas
se seleccionaron para su posterior identificación por espectrometría de masas.
Las MNT entre ellas resultó en un 80% de proteínas comunes en al menos dos cepas,
misma que disminuyó hasta el 17% con tres ó mas comparaciones, al incluir a M.
tuberculosis este resultado disminuyó a sólo el 4%.
De las proteínas comunes identificadas se encontraron 6 de metabolismo y cuatro mas
que participan en mecanismo de duplicación, transcripción y traducción. En cuanto a las
proteínas únicas para cada cepa de MNT están asociadas a virulencia y de función aun
desconocida.
Cuatro proteínas comunes a MNT y ausentes en el complejo tuberculosis son candidatas
de presencia y exposición a MNT.
41
L25
PROTEOME, PHOSPHOPROTEOME AND SECRETOME IN Rhizobium etli
1
Sergio Encarnación, 1Magdalena Hernández, 1Gabriel Martínez-Batallar,
1
Osbaldo Resendis, 1Sandra Contreras, 1Niurka Meneses, 2Guillermo Mendoza,
1
Yesenia Herrera, 1María del Carmen Vargas and 1Yolanda Mora
1
Centro de Ciencias Genómicas. Cuernavaca, México, 2Facultad de Medicina,
Universidad Nacional Autónoma de México, DF, México. Email:
[email protected]
PROTEOME
We are using proteomic approaches to determine the enzymatic reactions and
cellular processes that occur in free life, and in the symbiosis R. etli- Phaseolus
vulgaris. The identity of proteins in 1200 spots was determined. Using the
database of the R. etli genome and the software “Pathway tools” we constructed
“in silico” the potentially functional pathways and metabolic reactions
(Metabolome); we called this database Rhizocyc. Rhizocyc is composed of 45
pathways involved in many common anabolic and catabolic cellular processes of
small molecule metabolism which can be considered active in R.etli. A
comparative analysis of free-living and nodule residing bacteria revealed major
differences and similarities between the two states.
SECRETOME
This term is used to describe the proteome among all the secretions of the cell.
The proteomic application we used to study these proteins in each of the bacterium
growth phases was capable of answering many unknown questions about the
extracellular proteins’ functions. Two replicates of the secreted proteins from the
exponential and stationary phases were analyzed. The proteins were identified by
liquid chromatography coupled to mass spectrometry. The proteins were grouped
by COG (Cluster Orthologous Groups) according to NCBI. In the exponential
growth phase, 193 proteins were identified. It is interesting to note that we found
virtually all the machinery related to protein biosynthesis but we were unable to
identify the role they could play when secreted. A similar result was reported in E.
coli where they were suggested to be secreted in membrane vesicles; this led us to
investigate whether our bacterium also secretes external membrane vesicles. In
the stationary growth phase, 191 proteins were identified; we found that the largest
42
portion of these lack an associated function and they represent 28.7% from the
total of identified proteins.
PHOSPHOPROTEOME.
Protein phosphorylation is a post-translational modification that is widely
recognized as an important mechanism of signal transduction and regulation in the
living cell. The phosphoproteome is expected to be dynamic, responding to
different external and internal stimuli. Therefore, analyzing how the
phosphoproteome changes in different growth conditions is expected to provide a
deeper insight into the mechanisms and functionality of protein phosphorylation.
The functional distribution of phosphorylation sites in R. etli reveals that majority of
phosphorylated proteins have an enzymatic role. Interestingly, proteins encoded by
essential genes have been reported as overrepresented among the
phosphoproteins. Among the phosphorylated proteins approximately one quarter of
the enzymes are involved in pathways of carbon metabolism. Other prominent
groups include enzymes involved in DNA and protein metabolism, as well as stress
response. In addition, the R. etli phosphoproteome also contains a significant
portion of phosphorylated proteins with unknown functions. The subset of
phosphoenzymes involved in carbon metabolism seems to be “strategically”
distributed throughout various pathways, including both central pathways such as
glycolysis and the TCA cycle.
Part of this work was supported by CONACYT grant 60641 and DGAPA-PAPIIT
grant IN222707.
43
L26
HOST-PATHOGEN INTERACTIONS IN TUBERCULOSIS: A PROTEOMIC
APPROACH
1
Clara Inés Espitia Pinzón,2Gillermo Mendoza-Hernández, 1Wendy Xolalpa and
1
Margarita González-Zamorano
1
Departamento de Inmunología. Instituto de Investigaciones Biomédicas.
Universidad Nacional Autónoma de México. México, D.F. México. 2Departamento
de Bioquímica. Facultad de Medicina. Universidad Nacional Autónoma de México.
México, D.F. México. E-mail [email protected]
The interaction of bacteria with mammalian cells is a key process that allows
recognition, entry, invasion and persistence of many microorganisms in the host.
The human pathogen Mycobacterium tuberculosis binds a number of host cell
proteins including those of fibrinolytic system, Extracellular Matrix, complement,
mannose and DC sing receptors, among others. In order to identify the bacterial
ligands of some of these cell receptors, M. tuberculosis culture filtrate and cell
fractions were analyzed by two-dimensional electrophoresis combined with ligand
blotting. A protein fraction containing mannose enriched molecules was also
obtained by ConA affinity chromatography. Proteins were resolved by 2D-gel,
transferred to PDVF membranes and then incubated with plasminogen, fibronectin
and ConA. Several spots were detected and then identified via peptide mass
fingerprinting by a matrix assisted laser desorption/ionization time-of-flight mass
spectrometry.
With ConA, about 32 spots were detected and the majority corresponded to
lipoproteins with glycosylation sites predicted with NetOGlyc 3.1 software. In the
other wise several plasminogen and fibronectin binding spots were identifying and
corresponded to known proteins such us DnaK, GroES, GlnA1, Ag85 complex and
many other proteins that belong to metabolisms. Binding of plasminogen to
recombinant M. tuberculosis DnaK, GlnA1 and Ag85B was further confirmed by
ELISA and ligand blotting assays. The binding was inhibited by ε-aminocaproic
acid, indicating that the interaction involved lysine residues. Plasminogen bound to
recombinant mycobacterial proteins was activated to plasmin by tissue-type
plasminogen activator. In contrast with recombinant proteins, M. tuberculosis
soluble extract enhanced several times the plasminogen activation mediated by the
activator. Together these results show that M. tuberculosis posses a variety of
ligands that could interact with mammalian cell receptors, endowing bacteria with
different abilities that potentially contribute to establishment of the infection.
44
L27
A DATA INDEPENDENT APPROACH TOWARDS THE QUALITATIVE AND
QUANTITATIVE PROFILING OF COMPLEX PROTEIN MIXTURES
Johannes P.C. Vissers
Waters Corporation, MS Technologies Center, Manchester, UK
Mass spectrometry is widely accepted as an essential tool to better understand
protein function, facilitating both the identification and quantification of proteins in
complex samples. A mass spectrometry based protein identification strategy has
previously been described that facilitates the simultaneous acquisition of qualitative
and quantitative information, in a data independent fashion. This approach has
been extended to generate estimate amounts for proteins contained in biological
systems, allowing precise relative quantification to be performed.
The general challenges faced when analyzing complex protein mixtures and the
validation of the associated search results will be discussed in detail. The
specificity of both qualitative and quantitative analysis is in general lost when
chimeric peptides are considered and the effect that they have on the experiment
design, and outcome, is not acknowledged during either the acquisition or
searching of the data. It will be demonstrated through the use of experimental data
and modeling studies that the highest peak capacity afforded to detect, separate
and correlate all precursor and product ions by an analytical system is desired. The
latter is however not only being offered by separation in time, mobility and mass
but also through the use theoretical models of (sub) proteomes, considering
complexity, dynamic range and the inherent physiochemical properties of tryptic
peptides in both the solution and gas phase.
45
L28
RECENT DEVELOPMENTS IN MS/MS TECHNOLOGY
Jack Throck Watson
Emeritus Professor, Departments of Biochemistry and of Chemistry
former Director, MSU Mass Spectrometry Facilities, Michigan State University
East Lansing, MI 48824, e-mail: [email protected]
During the last two years, the performance specifications of commercially available
MS/MS instrumentation have improved dramatically. New standards of
performance at a resolving power of 60,000 and a spectrum generation rate of 20
Hz translates into more reliable protein identification and lower detection limits
during proteomic analyses by LC/MS. Dissociation of selected peptide/protein ions
by collisional activation (CAD), also called collision induced dissociation (CID), has
been complemented by the still developing techniques of electron capture
dissociation (ECD) and electron transfer dissociation (ETD); representative
examples will be described including one based on simultaneous ETD/CID. Ion
mobility spectrometry (IMS) has become a valuable technique for separating
gaseous ions based on differences in conformational shape or cross section,
including ions having the same m/z value. MS/MS instrumentation incorporating
IMS technology will be described in the context of separating families of protein
ions having the same m/z value, but different conformational shapes (and,
therefore, different drift times).
Keywords: Mass spectrometry, ETD, ECD, IMS, CAD, CID.
46
SESIONES ORALES
Salón Real San Luis “A”
15:30 – 16:30
47
L29
PROTEOMIC ANALISYS OF EFFECT PRODUCED BY FOCAL CEREBRAL
ISCHEMIA IN A RAT MODEL
Sánchez-Hernández H.1, Cázares-Raga FE.1, Cortés-Martínez L.1, HernándezHernández FC.1, Ortiz-Plata A.2
1
Depto. de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Av. IPN 2508,
Col. Sn Pedro Zacatenco, México, D.F. 07360, Tel. 57473800 Ext. 5646, E;
2
Instituto Nacional de Neurología y Neurocirugía MVS, Av. Insurgentes Sur 3877,
Col. La Fama, México, D.F. 14269, Tel. 56063822 x 2008.email:
[email protected]
Cerebral vascular disease (CVD) is a group of brain dysfunctions caused by blood
vessels failing, one of those is focal cerebral ischemia (FCI) that leads to reduced
blood supply in a specific zone of the brain, diminishing the glucose and oxygen.
FCI causes biochemical and protein expression changes in glial cells and neurons
resulting in enhancing or diminishing the ischemic injury. CVD is the third cause of
mortality in USA and fifth in Mexico. CVD can cause cerebral infarction and in this
case it is the first cause of disability and second of mortality in the world. Our
objective was to study protein expression in three different cerebral regions after
FCI experimentally induced in a rat model. We analyzed the hippocampal (H),
striate body (SB) and parietal cortex (PC) proteins after 1 h of ischemia and with or
without 24 h of sanguineous reperfusion (rpf), comparing with appropriate controls,
by two dimensional gel electrophoresis. The images were analyzed with Image
Master 2D Platinum 6.0 software for identification of differentially expressed
proteins. We found 57 shared proteins among the three cerebral regions in normal
conditions. Under 1 h FCI we observed 3 proteins that rise only in SB and 7
specific of PC in both conditions with and without 24 h of rpf. In addition, a specific
protein rose in H with 1 h of FCI without rpf. All spots will be identified by MALDITOF/MS and cell processes activated in response to injure will be suggested.
Key words: Cerebral ischemia, sanguineous reperfusion, differentially expressed
proteins
48
L30
DIFFERENTIAL PROTEOMIC ANALYSIS IN LIVER OF DIABETIC DB/DB MICE
Flores-Pérez Elsa C1, Mares-Álvarez Daniela P1, Flores-Altamirano Fátima1,
Ramírez-Emiliano Joel1, López-Briones Sergio1, Encarnación Sergio2 y PérezVázquez Victoriano1
1
Departamento de Ciencias Médicas, Universidad de Guanajuato, Campus León.
México. 2Centro de Ciencias genómicas, UNAM. E-mail: [email protected].
Type 2 diabetes mellitus is a public health problem in México and the world.
Diabetes mellitus is associated with metabolic and cardiovascular complications
such as glucose intolerance, dyslipidemia, coronary heart disease and
nonalcoholic fatty liver disease, which may eventually lead to hepatic fibrosis and
liver failure if left untreated in patients with type 2 diabetes. So, diabetes is a
chronic, complex, metabolic disorder affecting multiple organs such as kidneys,
eyes, heart and liver. We performed proteomic analysis to screen for global change
of hepatic protein expression in diabetic liver. Protein extracted from the liver of
ten-weeks-old db/db and non-diabetic mice were separated by two-dimensional
polyacrylamide gel electrophoresis and visualized by coomassie and silver staining
(n=4 in each group). Quantitative intensity analysis revealed 36 protein spots
differentially expressed in the liver from diabetic vs. control mice, of which 14
disappear and 4 diminished their expression. Additionally 10 spots appear of novo
and 8 increased their expression in diabetic mice liver. Ours results suggest
change in protein expression induced by hyperglycemia and diabetes
complications. It will be important identified the proteins with differential expression
in order to find the possible functional relevance with diabetes type 2.
Acknowledgements. The authors appreciated the financial support of CONCYTEG
(09-16-K662-079 NUM 2) y PROMEP-SEP. EFP is CONACYT fellow. We thank
M.Sc. A. Luna-Rocha and Sr. L. Galvan for helping us with housing mice.
49
L31
POTENTIAL BIOMARKER IN BREAST CANCER DERIVED FROM PROTEOMIC
ANALYSIS: BIOCHEMICAL CARACTERIZATION OF PYRUVATE-KINASE M2
(PK-M2)
Díaz-Tufinio, C, Cruz-Colín, JL, Gallegos-Pérez JL, Calderón-González K,
Gutiérrez-Nájera, N
Medical Proteomics Facility, National Institute of Genomic Medicine, Mexico. Email: [email protected]
Breast cancer is one of the main feminine death causes in Mexico. Due to its
frequency and gravity, it is necessary to develop fast, sensitive and specific
diagnosis methods. Through the systematic study of proteomics in pathologic
metabolic conditions, a better molecular comprehension of the disease can assure
accurate diagnosis techniques and treatments that promise better life quality for
the patient.
In this study, after a comparative proteomic analysis in differential two-dimensional
gel electrophoresis (DIGE) of three pure breast cancer cell lines (MCF7, Hs578t,
MDA-MB231 from ATCC), we identified pyruvate-kinase M2 isozyme (PK-M2).
Because of its frequency and abundance, we performed the biochemical
characterization of this enzyme for subsequent analysis in breast cancer patients.
We achieve yields greater than 80% of the enzyme in chromatography purification
steps, as well as fractions with purity factors about 50. Furthermore, in each
enzymatic activity determination we obtain one or two fractions with NADH
degradation rates higher than the rest of them. Moreover, with the addition of
fructose-1,6-diphosphate (F-1,6-dP), we induced a worthy increase of the activity,
proving the effect of PK-M2 tetramerization and an increased enzymatic activity in
presence of high F-1,6-dP concentration, a common molecular mechanism in
advanced cancer stages.
The perspective of the project is to try the same protocol in tumoral breast tissue
biopsies and in serum and urine due to the soluble nature of PK-M2, to quantify
and determine its enzymatic activity. These biochemical studies will be done to
offer a better diagnostic impression of aggressiveness and evolution of cancer.
50
L32
DIFFERENTIAL PROTEIN EXPRESSION IN HUMAN PTERYGIUM
Bautista-de Lucio VM1, Garfias Y1,2, López-Espinosa NL1, Zenteno E3, Mendoza
G3.
1
Unidad de Investigación, Instituto de Oftalmología “Conde de Valenciana”,
México D.F., 2 Departamento de Inmunología, Instituto de Oftalmología “Conde de
Valenciana”, México D.F., 3 Departamento de Bioquímica, Facultad de Medicina,
UNAM, México D.F. E-mail: [email protected]
Pterygium is one of the most frequent pathology in ophthalmology, and is a bening,
fibrovascular lesion, originated from the bulbar conjunctiva. It is composed of
epithelium and highly vascular, subepithelial, loose connective tissue. The
ethiology of pterygium is not clearly understood; the most widely recognized origin
factor is ultraviolet radiation. It has been proposed that pterygium and neoplasia
have common features raising the possibility that pterygium is a neoplastic-like
growth disorder. The aim of this study was to investigate the differences in protein
expression between pterygium and healthy conjunctiva. Four pterygium specimens
were obtained together with healthy conjunctival tissue from the same eyes. Total
proteins of pterygium and conjunctiva were analyzed in SDS-PAGE. This analysis
showed protein bands expressed exclusively in pterygium samples at the range of
20-25 kDa. After that, two-dimensional electrophoresis was performed for the
separation of total proteins; differential spots expressed in pterygium were
identified and selected, to be analyzed by matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. These
proteins were identified as peroxiredoxin 2, apolipoprotein AI and
proapolipoprotein. Peroxiredoxin-2 protects the cell against oxidative stressinduced apoptosis, and apolipoprotein AI has antiapoptotic effects and is related
with carcinogenesis and progression. These results support that pterygium is a
neoplastic-like growth disorder.
51
L33
AN IMMUNOPROTEOMIC APPROACH TO IDENTIFY POTENTIAL
BIOMARKERS FOR Trichomonas vaginalis
L.A. Ramón-Luing1, F.J. Rendón-Gandarilla1, R.E. Cárdenas-Guerra2, J. OrtegaLópez2, L. Avila-González1, C. Angel-Ortiz1, and R. Arroyo1
1
Departamento de Infectómica y Patogénesis Molecular, and 2Departamento de
Biotecnología y Bioingeniería, CINVESTAV-IPN, Mexico City, Mexico. Email:
[email protected]. Grants: 58611 and 68949 (CONACYT), and from ICyTDF.
Trichomonas vaginalis is a protozoan parasite responsible of trichomonosis, a
sexually transmitted infection. This parasite possesses multiple proteinases mainly
of the cysteine type (CP). In vivo some CPs are found in vaginal secretions of
patients with trichomonosis confirmed by in vitro culture, the diagnosis gold
standard method. Additionally, parasite-specific antibodies against CPs are
detected in these patient sera. Although, trichomonosis can be diagnosed by
different methods, a major problem is that ∼50% of the infected population is
asymptomatic. Therefore, a challenge is to design an alternative, specific, highly
sensitive, and inexpensive immunodiagnostic method based on T. vaginalis
antigenic proteinases. The goal of this study was to identify antigenic trichomonad
proteinases and pre-validate them as potential biomarkers for serological detection
of patients with trichomonosis. By an immunoproteomic approach using serum
from four T. vaginalis culture-positive patients, the major immunoreactive spots
identified were cysteine proteinases (TvCP2, TvCP4, TvCP4-like, TvCPT, and
TvLEGU-1). The genes of three CPs (TvCP4, TvCPT, and TvLEGU-1) were cloned
and expressed in Escherichia coli. Purified recombinant CPs were used as
antigens in Western blot assays to pre-validate them as potential biomarkers using
8 Tv (+) and 2 Tv(-) patient sera. Our results show that these CPs are indeed
biomarkers that could be used in ELISA-based assays, for the diagnosis of active
trichomonosis in asymptomatic patients or patients with vaginitis or urethritis.
52
L34
ENRICHMENT OF PHOSPHOPEPTIDES BY METAL ORGANIC AFFINITY
CHROMATOGRAPHY (MOAC) USING TIO2/NUTIP
Y. Herrera-Salgado, S. Contreras, M. Elizalde, A. G. Martínez, M. Hernández and
Sergio Encarnación.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México.
We describe a new efficient approach to analyze phosphopeptides in unique
separated protein from two-dimensional gel electrophoresis. In this method, a
titanium dioxide (TiO2)-packed NuTip is used as a phosphopeptide trap in which
beads are packed in a 20-µl pipet tip. Here, we present a suitable technique to
enrich in a more efficiently way the phosphopeptides, optimizing the time of
adsorption of phosphate groups on the surface of beads-Titanio in each step of the
sample enrichment. The use of displacers as DHB and lactic acid in the loading
buffer was used to improve the selectivity of enrichment. The results were obtained
from the comparison of mass spectra of proteolytic peptides from proteins without
and TiO2 enriched, determined by the gain of 80 Da in its sequence, using a
MALDI-TOF instrument. We have applied this method to corroborate the
phosphorylation of nine identified proteins with Pro-Q Diamond stained involved in
the symbiosis of Rhizobium etli with Phaseolus vulgaris.
grant IN222707
53
L35
PHOSPHOPEPTIDE ANALYSIS IN Arabidopsis thaliana SUBJECTED TO HIGH
LIGHT GROWTH CONDITIONS
Barba de la Rosa APab, Reiland Sonjaa, Bishof Sylvaina, Roschitzki Berndc,
Gerrits Bertranc, Baginsky Sachaa
a
Institute for Scientific and Technological Research in San Luis Potosi, México.
b
Plant Biotechnology, ETH-Zurich, Switzerland, cFunctional Genomic Center
Zurich, Switzerland cMolecular. E-mail: [email protected];
[email protected]
Protein phosphorylation plays a crucial role in almost all cellular and developmental
processes including signal transduction, translocation, and protein-protein
interaction. Mass spectrometry is one of the most powerful techniques for protein
identification, and characterization of post-translational modifications, including
protein phosphorylation. However, because phosphopeptides are often of low
abundance selective enrichment steps are required.
A novel, typically prokaryotic, sensor kinase in chloroplasts has been characterized
in Arabidopsis thaliana. The CSK protein is synthesized in the cytosol of
photosynthetic eukaryotes and imported into their chloroplasts as a protein
precursor. CSK provides a redox regulatory mechanism that couples
photosynthesis to gene expression. The aim of this work was to analyze the
changes in phosphoproteins in wild type A. thaliana and two T-DNA insertion lines,
Salk lines 027360 and 018074 where the gene At1g67840 encoding for the CSK
protein was disrupted. Arabidopsis thaliana seedlings were grown from seeds on
soil at 24°C and a photon flux density of 100 µEm-2 s-1 with an 8-hour light and 16h dark photoperiod. For high light experiments plants were grown in white high light
intensity (920 µEm-2 s-1) for 2 h. Leaves from 10 to15 plants were collected for
DNA, RNA, and protein extraction for plant genotyping and phosphopeptide
analysis. Total soluble and membranes proteins were digested with trypsin, and
phosphopeptide enrichment was achieved by strong cation exchange
chromatography (SCX), IMAC and TiO2 chromatography. Phosphopeptides were
analyzed and identified with an LTQ-Orbitrap mass spectrometer (Thermo Fischer
Scientific) interfaced with a nanoelectrospray ion source. MS and MS/MS data
were searched against the Arabidopsis database using MASCOT 2.1.04 (Matrix
Science) and INSPECT version 14.10.2008. Beside carbamylation of Cys residues
as a fixed modification, oxidation of Met and phosphorylation of Ser, Thr, and Tyr
were included as variable modifications. With MASCOT, database searches were
restricted to tryptic peptides, missing maximal two cleavage sites and protein Cand N-terminal peptides, allowing for 2+ and 3+ charged peptides a parent mass
error tolerance of 5 ppm and a daughter ion error tolerance of 0.6 D. The analyses
of phosphopeptides are discussed.
AP Barba thanks to Sabbatical Fellowship-CONACyT.
54
L36
PROTEOMIC OF PRICKLY PEARS WITH CONTRASTING RIPENING
BEHAVIOR
Rosas-Cárdenas F.F.1, Paredes-López O.1 , Cruz-Hernández A.1
1
Depto de Biotecnología y Bioquímica, Centro de Investigación y de Estudios
Avanzados de IPN, Unidad Irapuato. 36500, Irapuato Gto., México. Email:
[email protected]
The proteomics has been used to identify proteins involved in several biological
processes including fruit ripening. In this work we analyze the protein differential
expression and identify the proteins associated to the ripening of prickly pears with
different behavior (of early, intermediate, and late ripening). Fruits from different
morphospecies were harvested at green, middle-ripe and ripe stages. The proteins
were extracted from the peel and resolved on 2-DE gel, analyzed with the Image
master 2D Platinum 6.0 software (Amersham Biosciences) and grouped in Venn
diagrams. Differential spots were analyzed by spectrometry mass approaches. The
analysis showed a differential expression and a high synthesis of proteins during
ripening, the highest differential expression was obtained at the ripe stage of all
materials. 1689 proteins were associated to the ripening process. We identified
proteins associated with fruit ripening and with specific activities, such as
photosynthesis, respiration, fatty acids and anthocyanins synthesis. This
investigation provides the first proteomic approach for ripening of prickly pear fruits
with contrasting characteristic and allowed to analyze the great diversity of
peptides and the proteins that could be key to understanding and subsequent
practical application for control of the ripening in this important fruit.
55
L37
PROTEOMIC ANALYSIS OF AMARANTH UNDER DROUGHT STRESS
Huerta-Ocampo José Ángel1, Mendoza-Hernández Guillermo2, De LeónRodríguez Antonio1, Barba de la Rosa Ana Paulina1
1
Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí,
México.
2
Facultad de Medicina, Universidad Nacional Autónoma de México, Médico D.F.,
México. Email: [email protected]
Amaranth is a crop considered as drought-tolerant specie, it has been observed to
withstand drought stress better than wheat, maize, sorghum and cotton. Amaranth
seeds have high-protein content seeds with high lysine content, unique starch and
oil characteristics, biopeptides with antihypertensive and anticarcinogenic activity,
and several secondary metabolites with medicinal properties. The aim of this work
was to apply the comparative proteomics approach to study the differential
expression of amaranth roots and leaves proteins under drought stress. Twodimensional electrophoresis protein profiles were obtained. Drought-responsive
proteins were identified by tandem mass spectrometry. The results show that
stress-responsive proteins in amaranth leaves are mainly focused in stabilization of
other proteins. Decreased of Rubisco large subunit, oxygen evolving and
cytochrome b6f complex and the enzyme fructose-bisphosphate aldolase shows
that the effect of drought on electron transport chain could be related with the
reduction of carbon metabolism. Amaranth root adaptation to drought stress is a
coordinated pathway of proteins that control the damage from reactive oxygen
species, a family of heat shock proteins that stabilize other proteins, and the downregulation proteins involved with lignification seems to be the adaptive responses
to severe drought. These results open the door for further investigation, which will
lead to a better understanding of the tolerance to abiotic stress in amaranth.
This work was supported by the European Commission 6th Framework
Programme, AMARANTH: FUTURE-FOOD, Contract No. 032263
56
L38
DIFFERENTIAL PROTEIN EXPRESSION IN ANOPHELES ALBIMANUS
MIDGUT INFECTED WITH PLASMODIUM BERGUEI
Vania V. Serrano-Pinto1,*, Maribel Acosta-Pérez, M.2, Darwin Luviano-Bazán2,
Gerardo Hurtado-Sil2, Cesar V. F. Batista3, Jesús Martínez-Barnetche2 and
Humberto Lánz-Mendoza2.
1
Centro de Investigaciones Biológicas del Noroeste, Mar Bermejo 195, Col. Playa
Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico. 2Instituto Nacional de Salud
Pública, Centro de Investigaciones Sobre Enfermedades Infecciosas. Av.
Universidad 655, Col Santa María Ahuacatitlán, Cuernavaca, Morelos 62100,
Mexico. 3Unidad de Proteómica-Instituto de Biotecnología, Universidad Nacional
Autónoma de México (UNAM), Av. Universidad 2001, Col Chamilpa, Cuernavaca,
Morelos 62210, Mexico. Email: [email protected]
The Malaria is one of the most important illness in tropical and subtropical
countries around the world. The mosquito Anopheles is the main vector of the
human Malaria parasite Plasmodium. The Malaria infects 300 millions of people
around the world and kills more than a million each year. In the Americas, high
rates of morbidity due to Malaria infection are reported every year. In Mexico are
reported more than 16,000 cases, increasing the infection each year. The main
vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito.
The midgut of the Anopheles mosquito is the first blood reservoir after feeding and
is the first place of attack against the Plasmodium parasite; hence the importance
of a detailed functional analysis to determine the role of proteins in the gut in
eliminating the parasite.
In this study, we analyzed the midgut of An. albimanus infected with P. berghei.
We used a proteomic approach to identify proteins that are enriched in the midgut
after blood feeding. The mosquito’s midgut were analyzed by two-dimensional
electrophoresis to determine the changes in protein profiles. We identified 21 spot
proteins that are differentially expressed in mosquitoes after blood feeding with P.
berghei. Molecular weight of the spots varied between 13 and 36 kilodaltons (kDa),
with a large isoelectric point (Ip) range of 3.92 to 8.90. Proteins differentially
expressed were identify using mass spectrometry. A proteome data base of An.
albimanus midgut was obtained and information of several proteins with digestion
and immunological function was documented. Some of these proteins may have
important implications for understanding the blood meal digestion process and the
parasite vector interaction.
57
L39
IDENTIFICATION OF IMMUNOGENIC PROTEINS FROM Mannheimia
haemolytica BY IMMUNOPROTEOMIC APROACH.
Patricia Ramírez R, Gonzalo Mendoza O. y Rodolfo Hernández G.
Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de
Jalisco, CIATEJ. Guadalajara, México. Email: [email protected]
The Bovine Respiratory Disease (BRD) is responsible for significant economic
losses in the feedlot cattle industry and Mannheimia haemolytica (Mh) is the
predominant etiological agent. Considering the difficulty of an adequate diagnosis,
and the need for an improvement of immunogenic proteins of Mh the identified
proteins will be potential candidates in the design of a chip for the diagnosis in field
and as new unitary vaccines or to upgrade commercial vaccines. Three
immunogenic proteins were identified out of two-dimensional western blots of 30
anti-sera (cattle immunized with Mh). The Proteins identified were analyzed by
mass spectrometry (MALdi-TOF MS). NP.9 (32.5 kDa) was identified as yfeA, a
Substrate Binding Protein (SBP) element of the ATP Binding Cassette (ABC)
transmembrane transport system, specifically transporter of Fe+ and of the TroALike superfamily. The protein NP.7 (55.5 kDa) was also identified as SBP of the
ABC transport system. The exact function of NP.8 (40.5kDa) is unknown but,
according to the National Center for Biotechnology Information (NCBI) database, is
probably a capsular biosynthesis protein. The immunological protection capability
of proteins of ABC system, of several pathogenic bacteria, has been demonstrated
experimentally in animals and many ABC systems have been considered as
virulence factors. Thus we propose yfeA (NP.9) and NP.7 as potential candidates
to be evaluated as vaccines. Nevertheless, their great homology with proteins of
many other bacteria limits their potential as antigens for a diagnosis chip. In
contrast, NP.8 doesn´t share homology with many bacteria and possesses
antigens particular of Mh, thus we propose it as good candidate to be evaluated for
a diagnosis chip.
58
L40
PHOSPHOPROTEOME ANALYSIS OF THE Saccharomyces cerevisiae IN THE
ANSWER TO OXIDATIVE STRESS.
Martinez Obregon F.1, 2, Herrera Salgado Y.2, Contreras S.2 and Encarnación
Guevara S.2
1
The Biotechnology Institute, Universidad Nacional Autonoma de Mexico,
Cuernavaca, Mexico, 2Genomics Sciences Center, Universidad Nacional
Autonoma de Mexico, Cuernavaca. Email: [email protected]
The oxidative stress is defined as the intracellular redox imbalance caused by the
reactive oxygen species over production and the incapacity of an antioxidant
system to eliminate them. The phosphorylation is the most common and important
mechanism for the precise and reversible regulation in the proteic function. The
global analysis of the phosphorylated proteins in a cell, in a determined moment
and condition, is the phosphoproteome goal. Due to the complexity of the
phosphorylation proteins patterns, a comprehensible analysis could be obtained
using different experimental strategies lead to the selective enrichment of the
phosphorylated proteins. The Saccharomyces cerevisiae oxidative stress
induction, was performed in an exponential growth medium stage, using the wildtype strain BY4741 [MATa his3∆1 leu2∆0 met15∆0 ura3∆0] and its mutant in
∆Skn7. Total protein was extracted after 3 hours of induction. Enrichment of
phosphorylated proteins was performed using MOAC [Metal Oxide Affinity
Chromatography], followed by two-dimension analysis. First, phosphoproteins were
separated using electrophoresis SDS-PAGE [Sodium-Dodecy Sulphate
Polyacrilamide Gel Electrophoresis] and were identificated using Nano LC-ESI
MS/MS [Liquid Chromatography coupled to tandem Mass Spectrometry with
Electrospray Ionization] finally. The partial bioinformatic analysis suggests an
important presence of a phosphostimulon when the yeast is found in the oxidative
stress.
Part of this work was supported by CONACyT grant 60641 and DGAPA-PAPIIT
grant in 222707.
59
L41
TWO-DIMENSIONAL BLUE NATIVE/SDS GEL ELECTROPHORESIS OF
Candida albicans MULTIPROTEIN COMPLEXES
J. Luis Martínez Salgado1, Aida Pitarch2, Ana Paulina Barba de la Rosa1 and
Concha Gil2
1
Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí,
México.
2
Departamento de Microbiología II, Facultad de Farmacia, Universidad
Complutense de Madrid, España. Email: [email protected]
Multiprotein complexes play a key role in many cell biological processes.
Their characterization is a first step towards further studies on protein-protein
interactions. We standardized the two-dimensional blue native-SDS polyacrylamide
gel (2D-BN/SDS-PAGE) technique using cytoplasmic extracts of opportunistic
pathogen Candida albicans. The yeast cells were disrupted with glass beads under
native conditions. To enhance the multiprotein complex resolution, it was essential
to perform several desalting steps before 2D-BN-PAGE. The desalted protein
extract was then run in a blue native 5-9% gradient gel using a Protean II
electrophoresis system. After that, the BN-PAGE lane was excised from the firstdimension gel and then equilibrated to reduce and alkylate the multiprotein
complexes. For the second dimension, this lane was subsequently separated in a
7.5-11% gradient gel under denaturing conditions. The resulting 2D-BN/SDSPAGE gel was silver-stained. Several biological and technical replicates were
carried out, confirming a good reproducibility of this method. Protein identification
was performed by MALDI-TOF mass spectrometry analysis. We identified eleven
proteins, including two subunits of the isocitrate dehydrogenase, grouped into three
multiprotein complexes and related to different central metabolic pathways. This
proteomic approach opens an attractive way towards the characterization of
important protein-protein interactions occurring during crucial metabolic processes
for this human fungal pathogen as well as towards future functional proteomic
investigations.
60
L42
IDENTIFICATION OF THE SUBPROTEOME OF Trichomonas vaginalis ACTIVE
ENDOPEPTIDASES: THE DIFFERENTIAL EXPRESSION OF PROTEINASES
BY IRON OR CELL CONTACT
L.A. Ramón-Luing, F.J. Rendón-Gandarilla, L. Ávila-González, and R. Arroyo
1
Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Mexico
City, Mexico. Email: [email protected]. Grants: 58611 and 68949
(CONACYT), and from ICyTDF.
Trichomonas vaginalis has many proteinases of the cysteine type (CP) involved in
pathogenesis. In the T. vaginalis genome 220 genes encoding CPs were found; by
two-dimensional (2-D) substrate gel electrophoresis (zymogram), up to 23 spots
with proteolytic activity have been detected. Our goal was to obtain and identify the
protein spot pattern of the active proteinases (degradome) and their differential
expression induced by iron or cell contact, using 2-D gel electrophoresis, 2-Dzymograms, and mass spectrometry (MS) approaches. Although the inherent
difficulties to work with active proteinases, we identified the degradome from
normal grown parasites; 41 silver-stained spots were detected in the region of 20010 kDa. Interestingly, a similar proteolytic pattern was observed in the 2-D
zymogram, suggesting that most of the silver-stained spots could correspond to
proteinases. This hypothesis was confirmed by MS. From the 27 identified proteins
spots, 21 corresponded to 9 distinct CPs (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4like, TvCP12, TvCPT, TvLEGU-1, and another TVLEGU). Noteworthy, some spots
with distinct molecular weight, but similar pI were identified as the same CP. The
differential expression observed in some CPs from parasites grown in distinct iron
concentrations is in agreement with the effect of iron at the transcriptional level for
some CP genes. Additionally, some CPs were differentially expressed in parasites
grown in contact with fixed HeLa cells. At least two novel spots were observed. Our
data suggest that T. vaginalis only expresses a small set of CP genes under the
studied conditions.
61
RESÚMENES DE PRESENTACIONES EN
CARTEL
Salón Real San Luis “B”
62
P01
PROTEOMIC ANALYSIS OF NASAL SECRETIONS FROM CHILDREN
SUFFERING PARAINFLUENZA VIRUS INFECTION
L. H. Gutiérrez-González1, J. Langridge2, T. McKenna2, L. M. Terán1
1
Instituto Nacional de Enfermedades Respiratorias, México, D. F., México,
2
Waters, Manchester, UK, Email: [email protected]
Human parinfluenza viruses (HPIV-1 to -3) are major cause of respiratory
infections in infants and young children. In the present study we have investigated
the proteomic profile in the nasal secretion of children developing naturally
acquired parainfluenza infection. METHODS: Nasal aspirate samples were taken
from children aged 7 - 12 years in the Mexico City area during acute naturally
acquired parainfluenza disease. Control samples were taken when the same
children had been asymptomatic for at least 4 weeks. Parainfluenza virus detection
was performed by immunofluorescence in nasal aspirate cells using an
immunofluorescence kit (Chemicon, 3105). Protein expression was analyzed using
bi-dimensional liquid chromatography followed by LC-MS (LC-MSE and DDA) in a
nanoACQUITY spectrometer (Waters, UK). RESULTS: 25 proteins have been
identified including surface molecules, extracellular matrix proteins, intracellular
signaling products, vascular proteins, and transcription factors which were not
present when the children were symptom-free. Our findings reveal that children
infected with parainfluenza virus secrete into their airways a number of activated
proinflammatory molecules that may be involved in the pathogenesis of
parainfluenza virus infection.
63
P02
PROTEIN BIOMARKERS USEFUL IN A DIFFERENTIAL SKIN CANCER
DIAGNOSTIC
Herrera-Carrillo Zazil1, Ramón-Gallegos Eva1.
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México,
D.F. E:mail: [email protected]
Epidemiological skin cancer it is divided in two groups: non-melanoma and
melanoma types Non-melanoma skin cancers comprise basal cell carcinomas
(BCC) and squamous cell carcinomas (SCC). In Mexico, BCC and SCC has the
first and third place in malignant skin cancer. The global incidence of malignant
melanoma (MM) and non-melanoma cancers continues to increase; however, the
main factors that predispose to the development of melanoma seem to be
connected with recreational exposure to the sun and a history of sunburn.
Nowadays the different methods to diagnose skin cancer are based in methods
that depend of observation and that not always allow a good diagnostic in the
primary steps of the cancer. That is why is necessary a novel methods based in
molecular biology and proteomic to diagnose a different skin cancers in a specific
way. The objectives of this work are find protein biomarkers useful for differential
diagnostic of skin cancer and propose a diagnostic method based in those
biomarkers. In general, the methodology consist in obtain proteins from formalinfixed paraffin-embedded tissues (FFPE) of normal skin (NS),MM, BCC, SCC and
nevo carcinoma (NC). These proteins will be processed to use in a protein analysis
by liquid chromatography-mass spectrometry (LC-MS/MS) and bidimentional
electrophoresis. Protein identification and classification in molecular function and
cellular localization will be determined by bioinformatics. A microarray will be
design with the protein biomarkers chosen like a novel diagnostic method for
differential skin cancer. Our study will provide useful information about a variety of
proteins which could be involved in benign and malignant skin cancer; also these
proteins could be use as a diagnostic marker for each type of tumor analyzed.
Key words: proteomic,
electrophoresis
biomarker,
skin
cancer,
LC-MS,
bidimentional
Preference: POSTER PRESENTATION
64
P03
ANALYSIS OF THE GLOBAL PATTERNS OF PROTEIN EXPRESSION IN
CELLULAR LINES OF CERVICAL UTERINE CANCER
Juan Carlos Higareda Almaraz1, Magdalena Hernandez Ortiz1, Sergio Encarnación
1
1
Centro de Ciencias Genómicas. Cuernavaca, México. Email: [email protected]
The cervical cancer, (CaCu) is the second cause of death associated with
neoplasia in the Mexican feminine population. Recent works suggest that during
carcinogenesis key processes are deregulated. Generally, development of
independence to growth factors, loss of sensitivity to factors of inhibition, evasion
of apoptosis, expansion of replicative capacity, induction of angiogenesis,
development of the invasion faculty and metastasis are the nucleus of this system
We explored the global expression patterns of six cell lines of cervical cancer
(HeLa, CaLo, SiHa, CasKi, ViBo and C33-A) using two-dimensional
polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Protein
expression was evaluated using PDQuest 2-D software and the peptide mass
spectra identification was performed using the Mascot program searching the
Swiss-prot or NCBInr databases.
74 electrophoretic entities were characterized, all of which have been reported to
participate in carcinogenic models, directly or indirectly. Finally, we used these
data to build a bioinformatic model that predicts possible novel interactions.
Part of this work was supported by CONACyT grant 60641 and DGPA-PAPiiT
grant IN222707.
65
P04
PROTEOMIC ANALYSIS IDENTIFIED GLIOXALASE I AS A OVEREXPRESSED
PROTEIN IN BREAST CANCER FROM MEXICAN PATIENTS
Miguel Á. Fonseca Sánchez1, Sergio Rodríguez Cuevas2, Elizbeth Álvarez
Sánchez1, Juan P. Luna3, Guillermo Mendoza4, César López-Camarillo1*
1
Universidad Autónoma de la Ciudad de México, Posgrado en Ciencias
Genómicas, México DF. 2Instituto de Enfermedades de la Mama, FUCAM.
3
CINVESTAV, Biología Celular. 4UNAM, Facultad de Medicina.
*Corresponding author: [email protected]
Breast cancer is the leading malignancy of women. Worldwide, over 1.5 million
new cases of breast cancer will be detected each year, and majority of deaths
cases will be occurs in developing countries. Remarkably, a new breast case is
diagnosed every 2 hours in Mexico. Highest mortality is because cancer is
detected in advanced stages of disease, which reduces life expectancy. The
mortality reduction requires developing molecular strategies for early detection and
appropriate therapy protocols ad hoc to molecular classification of tumors, which
may contribute significantly to patient survival and outcome.
In this work, we focused in the search of differentially expressed proteins in
sporadic breast tumors from Mexican patients. By using 2D-PAGE and proteomic
tools, we analyzed the protein expression profiles from healthy mammary tissues
and 10 breast tumors obtained from FUCAM. Our results showed that at least 9
proteins were overexpressed in breast tumors, whereas 15 were down-regulated.
One of the most abundant spots was excised from 2D-gels and identified by LCMS/MS mass spectrometry. Identified protein corresponds to glyoxalase I (GLOI),
which functions in the detoxification of methylglyoxal, a side product of glycolysis.
Accumulation of methylglyoxal causes DNA modification and protein cross-links
and activates apoptosis. Because, GLOI was only detected in breast tumors, it may
be a potential tumor marker candidate. Validation experiments, in progress, could
help us to define the precise role of GLOI in breast cancer.
66
P05
NEGATIVE EFFECT OF IRON IN THE PROCESSING OF THE Trichomonas
vaginalis CYSTEINE PROTEINASE TVLEGU-1.
F.J. Rendón-Gandarilla, L.A. Ramón-Luing, and R. Arroyo.
Departamento de Infectómica y Patogénesis Molecular. CINVESTAV-IPN. Email
[email protected]; [email protected]: 58611 and 68949
(CONACYT) and from ICyTDF.
The tvlegu-1 gene encodes for a ~42.8 kDa Trichomonas vaginalis precursor
cysteine proteinase of the legumain type of family C13 of clan CD, named
TVLEGU-1. Its expression is up-regulated by iron at the transcription and protein
levels. In spite of lacking transmembranal domains TVLEGU-1 is located on the
parasite surface. Its surface location is also regulated by iron. Moreover, by
Western blot (WB) assays, antibodies against a synthetic peptide or to the
recombinant TVLEGU-1 protein reacted with protein bands of 60, 50, and 30 kDa
in trichomonad extracts. These data suggested that these protein bands could
correspond to different TVLEGU-1 processing stages; from the precursor to the
mature enzyme, as occur in mammal and other parasite legumain-like CPs. To test
this hypothesis, the aim of this work was to study the processing stages of
TVLEGU-1 in parasites grown at different iron concentrations. By 1-D and 2-D gel
electrophoresis combined with WB assays and mass spectrometry (MS)
approaches; different stages of TVLEGU-1 processing were detected. In normal
conditions, the three protein bands and spots (60, 50, and 30 kDa), corresponding
to the precursors and mature enzyme were identified by MALDI-TOF/MS.
Moreover, the TVLEGU-1 precursor proteins (60 and 50 kDa) were mainly
observed in iron-rich conditions; while the mature protein (30 kDa) was favored in
iron-depleted conditions. Additionally, potential cleavage sites for activation of
TVLEGU-1 were also identified. Thus, our data show that the different processing
stages of TVLEGU-1 are privileged in the absence of iron.
67
P06
EVALUATION OF PARAFFIN-EMBEDDED TISSUES FOR BIOMARKER
DISCOVERY BY SHOTGUN METHODS
G.Y. Monroy Núñez2, J.P. Reyes Grajeda1, K. Calderón-González1, J.L. GallegosPérez1.
1
Unidad de Proteómica Médica. Instituto Nacional de Medicina Genómica,
INMEGEN.
2
Universidad Autónoma de Baja California (UABC).
[email protected]
A major focus in proteomics today is the search for biomarkers that accurately
indicate disease.
Once removed from patients, tissue specimens are typically either fixed in
aldehyde based fixatives (e.g., 10% formalin) or snap frozen. Although formalinfixed tissues are well preserved for histopathological evaluation, the quality of
macromolecules is severely compromised, thus hindering subsequent molecular
studies.
In this report, we analyze ethanol-fixed and formalin-fixed paraffin-embedded
(FFPE) samples of mouse liver using four different protocols for the protein/peptide
extraction and then analyzed by MS/MS.
68
P07
PROTEOMIC EXPRESSION MAP OF THE INTERACTION OF Trichomonas
vaginalis WITH PROSTATIC CELLS
Vázquez Carrillo, L. I1, Quintas Granados, L I1, Arroyo R2, Castañón Arreola, M1,
and Alvarez Sánchez M. E1.
1
Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de
México, D. F., México, 2Departamento de Infectómica y Patogénesis Molecular,
Centro de Investigación y de Estudios Avanzados Instituto Politécnico Nacional,
México, D. F. Email :[email protected].
Grants: 83808 (CONACYT), and from PIFUTP08-150 (ICyTDF)
Trichomonas vaginalis is the causative agent of trichomonosis, a sexuallytransmitted infection. In men, this infection is asymptomatic, but sometimes can
cause urethritis, chronic prostatitis, balanitis, epididymitis, and infertility. Prostatic
secretions contain high zinc concentrations (4.5 to 7 mM), which has antibacterial
and trichomonicidal activity. Zinc concentration lower than 1.6 mM is non
trichomonicidal and is found in patients with chronic prostatitis and prostate cancer.
The goal of this study was to investigate the zinc-dependent changes induced in
the proteome of T. vaginalis and its effect over the parasite interaction with cells
from a human prostate cancer line, called DU145. A comparative analysis by 2D
gel electrophoresis (2-DE) showed differences in the number of spots depending of
the zinc concentrations. These images were analyzed by the pDQuest software
and used for developing the proteomic expression map of T. vaginalis. We also
investigated the effect of zinc on the the cp65 gene expression by qRT-PCR the
CP65 proteolytic activity and the levels of trichomonal cytoadherence and
cytotoxicity. Our results showed that the levels of cp65 gene expression, proteolytic
activity, cytoadherence, and the CP65-dependent cytotoxicity were the lowest at
1.6 mM zinc concentration. In conclusion, we reported a proteomic expression map
of T. vaginalis grown in presence or absence of zinc and the negative effect of zinc
over the host cell-parasite interactions and its effect over the cp65 gene expression
in T. vaginalis.
69
P08
PROTEMIC ANALYSIS OF ZEBRAFISH EMBRYOS EXPOSED TO THE
ENDOCRINE DISRUPTOR 17-α
α-ETHINYL-ESTRADIOL
Teresa Gutiérrez Espíndola1,2, Ernesto Maldonado Olvera2, Karla Grisel Calderón
González3, Nora Andrea Gutiérrez-Nájera3, José Luis Gallegos Pérez3
1
CEACA,Facultad de Química, Universidad Autónoma de Querétaro, Querétaro,
México, 2Instituto de Fisiología Celular, UNAM.,México, D. F.,México, 3Instituto
Nacional de Medicina Genómica, México D.F., México. Email:
[email protected]
Contamination of water bodies such as lakes, rivers, seas, and aquifers by
Endocrine Disruptor Chemicals (EDCs) is a major concern in developed countries.
There is a general agreement about EDCs have a high impact on reproduction and
development in human and fauna. 17−α-ethinyl-estradiol (EE2), a synthetic steroid,
is one of the most commonly used oral contraceptive pills in Europe and America.
EE2 is excreted to the environment by urine and feces as a xenoestrogen of
people who use this synthetic steroidal estrogen. It has been observed that this
compound remains biologically active provoking feminization and other abnormal
endocrine responses in fish population. The objective of this work was to determine
if zebrafish embryos exposed to EE2 show changes in their proteomic expression.
Thus, this model organism could be useful as biological monitor for water sources
that could be potentially contaminated with EDCs. Difference Gel Electrophoresis
(DIGE), shotgun proteomics and Western Blot techniques where used to study the
protein expression differences between exposed and non-exposed embryos.
Preliminary results showed some changes between both proteomes. Vitellogenin,
a well known protein biomarker for exposure to environmental estrogens, was
observed to be induced in embryos exposed to a high concentration of EE2. These
results may lead future research to confirm the use of zebrafish to determine if
water sources can be considered “safe” to be used in human activities or as
potable water.
Keywords: Endocrine disruptors, 17-α-ethynil estradiol, zebrafish, proteomics,
mass spectrometry.
70
P09
PROTEOMIC ANALYSIS OF EFFECT OF PROSTAGLANDINS ON MIDGUTS
OF THE DENGUE VECTOR MOSQUITO Aedes aegypti
Borbolla-Vázquez J1., Cázares-Raga FE1., García-Gil de Muñoz FL2., SánchezHernández H1., Cortés-Martínez L1., Rodríguez MH3, Hernández-Hernández FC1.
1
Depto. de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, México, D.F.
2
Universidad Simón Bolívar México, D.F., 3CISEI-INSP, Cuernavaca, Morelos,
México. email: [email protected].
Dengue is an infectious disease produced by the dengue virus (DEN), which is
transmitted to the human by female Ae. aegypti. Currently DEN affects 50-100
million people in the world, In Mexico are reported up to week 37 of 2009, 16,237
and 3,583 cases of classic and haemorrhagic dengue. Mosquito ingest DEN during
blood meal, initially viruses reach the midgut and in subsequent days invade the
body including salivary glands and pass through next patient in saliva during
probing. Prostaglandins (PGs) are important mediators of humoral and cellular
immunity in the insects. The objective of present study was to analyze the effect of
PGE2 on protein expression of the midgut of Ae. aegypti. The protein analysis was
conducted by two dimensional electrophoresis, and the protein profiles compared
between experimental conditions using Image Master 2D Platinum. We observed
150 proteins spots in control; 120 spots with PGE2 and 130 spots with IBU.
Treatment with PGE2 induced a spot of 75 kDa and pI 5.9 and with IBU, an
inhibitor of PGE synthesis, two spots of 14 and 15 kDa with pI 6,8 were induced.
These results suggest that PGE2 regulate the proteins expression in Ae. aegypti
midguts. All spots will be identified by LC-MS/MS in order to know their function
because any of them could be related to the immune response this mosquito
vector.
Key words: prostaglandins, Ae aegypti, dengue.
71
P10
PROTEOMIC ANALYSIS OF CELL MEMBRANES OF MIDGUTS OF DENGUE
VECTOR Aedes aegypti
Garibay García JAa, Cázares-Raga FEa, Sierra-Martinez Pb, Rodríguez MHc,
Hernández-Hernández FCa.
a
Depto. de Infectómica y Patogénesis Molecular. CINVESTAV-IPN. Av. IPN 2508,
Sn Pedro Zacatenco. México, D.F. 07360. Tel: 57473341. bUnidad de
Investigación Especializada en Microbiología-UAG. cCISEI-INSP Cuernavaca Mor.
Email: [email protected]; [email protected]
The mosquito Aedes aegypti is the main vector of dengue. Blood feeding is a
female-specific habit used by infectious agents, such as viruses and parasites for
transmission. Cell membranes participate in homeostatic mechanisms in response
to environment; molecules associated to outer membranes are the first contact with
infectious agents and transduce signals toward cell inside. Inner membranes are
involved in cell responses including secretion and apoptosis between others.
Mosquito female midgut cells synthesize, in response to meal composition,
specialized proteins as enzymes and peritrophic matrix components but these
have not been fully described and it is necessary to establish which databasepredicted molecules are present. Objectives. Due to the importance of
membranes in immunity, development and bloodfeeding of the mosquito, in this
work we identify membrane proteins from the midgut of sugar-fed Ae. aegypti.
Methods. Midguts of sugar-fed adult females of Ae. aegypti were dissected,
ultracentrifugation-fractionated and membrane proteins resolved by SDS-PAGE.
Selected protein bands were excised and identified by ESI-LC-MS/MS. Results. In
membrane preparations of sugar fed midguts eight bands were enriched in
comparison with total crude extracts. Five bands were studied by MS, and 15
proteins identified corresponded to outer and inner membrane and mitochondrial
proteins, previously reported only as hypothetic proteins in mosquito databases.
Keywords: Aedes aegypti, midgut, proteomic analysis.
72
P11
NOVEL DERMASEPTIN-LIKE ANTIMICROBIAL PEPTIDES FROM THE
MEXICAN FROG PACHYMEDUSA DACNICOLOR: SEQUENCING BY
MULTIPLEX MASS SPECTROMETRY DISSOCIATION METHODS
Erika Patricia Menezes1, Oscar Villa Hernández1, Lorena Hernández Orihuela1,
Ruben Castro Franco2 and Cesar V. F. Batista1
1
Unidad de Proteómica, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Cuernavaca, México
2
Lab. Herpetología, Centro de Inv. Biológicas-UAEM, Cuernavaca, Mor-México.
E. mail: [email protected]
Skin secretion of amphibians is a rich source of bioactive peptides and its activities
have been demonstrated against bacteria, fungi, parasites and viruses.
Pachymedusa dacnicolor is an endemic Mexican frog that inhabits the southwest
region of the country. Skin secretion was obtained by electrical stimulation “in situ”,
immediately stored at –20°C and the animals release d to the field in healthy state.
After lyophilization, aliquots of 2 mg were separated by means analytical RPHPLC, dry in speedvac and analyzed by mass spectrometry. All HPLC fractions
were analyzed by an Orbitrap-FT mass spectrometer and a complete molecular
mass fingerprint was determined. The main fractions were further purified by flat
analytical HPLC gradients and the homogeneous components submitted to
fragmentation by ETD (Electron Transfer Dissociation), CID (Collision-Induced
Dissociation), HCD (High Energy Collision Dissociation) and PQD (Pulsed-Q
Dissociation) fragmentation methods. The analytical procedure adopted allows “de
novo” sequencing of dozens of enzymatic-generated peptides and full sequences,
as well. The components structurally characterized belong mainly to the
dermaseptin family and many peptides full sequenced possess the C-terminal
amidated. In conclusion, the use of multi- fragmentation methods is a powerful tool
for “de novo” sequencing, especially when CID and HCD are used in a
concomitantly fashion.
to CVFB.
73
P12
PROTEOMIC ANALYSIS OF THE SKIN SECRETION OF THE MEXICAN FROG
LITHOBATES PUSTOLOSUS
Oscar Villa-Hernández1, Ruben Castro Franco2, Cesar V. F. Batista1
1
Instituto de Biotecnología, Universidad Nacional Autónoma de México,
Cuernavaca, México, 2Lab. Herpetología de Biotecnología, Universidad Nacional
Autónoma de México, Cuernavaca, México, Email: [email protected]
Antimicrobial peptides (AMPs) are an essential part of innate immunity present in
most living organisms. These small cationic peptides are multifunctional as
effectors of innate immunity on skin and mucosal surfaces and have demonstrated
direct antimicrobial activity against various bacteria, viruses, fungi, and parasites.
Lithobates pustulosus is an endemic Mexican frog. To the best of our knowledge
the biochemical data contained herein is the first report describing the skin
secretion composition of this specie. Secretions were obtained by electrical
stimulation, immediately stored at –20°C and dried in a vacuum concentrator. Two
milligrams of secretion was dissolved in water, centrifuged and the supernatant
separated by RP-HPLC in an analytical C18 column. The main HPLC fractions
were repurified, digest with endoproteases and analyzed by an LTQ Orbitrap XL
mass spectrometer. Different dissociation methods were used to access the
primary structure. All spectra generated were manually analyzed and searched
against NCBInr Database. By this mean, many antimicrobial peptides were
identified, most of them belonging to brevinins, ranacyclins and ranatuerins
families. However, many peptides “de novo” sequenced are unknown sequences
that must be further studied at structural and pharmacological level.
to CVFB.
74
P13
CARACTERIZACIÓN DE LA RESPUESTA INMUNE HUMANA CONTRA
MICOBACTERIAS.
E. Amador-Gaytán, L. Lloret-Sánchez, A.I. Castillo-Rodal, Y. López-Vidal.
Programa de Inmunología Molecular Microbiana. Departamento de Microbiología y
Parasitología. Facultad de Medicina, UNAM. México D. F., Email:
[email protected]
Introducción. El género Mycobacterium incluye una gran variedad de especies,
desde saprófitas que viven en el medio ambiente, hasta parásitos obligados como
son M. leprae y los integrantes del complejo M. tuberculosis (CMT). Dentro de este
complejo M. tuberculosis destaca por ser el agente etiológico de la Tuberculosis
(TB) y M. bovis por ser la cepa de la cual se desarrolló la vacuna BCG. Esta
vacuna es usada ampliamente alrededor del mundo, presentando una eficiente
protección contra la TB en recién nacidos, pero no evita el establecimiento de la
TB latente o la reactivación de la TB pulmonar en adultos. Además del CMT, el
género Mycobacterium se compone de más de 150 especies denominadas como
micobacterias no tuberculosas (MNTs) que habitan una variedad de nichos que
comprenden fuentes de agua, suelo, aerosoles, protozoarios y animales que
incluyen al hombre. Las MNTs tienen el potencial de ser patógenas y algunas son
causantes de infecciones pulmonares con síntomas similares a la TB, lo que
dificulta su diagnóstico oportuno. Por lo tanto el desarrollo de herramientas de
diagnóstico específicas y sensibles permitirá detectar a los integrantes del CMT
por lo que el objetivo de este trabajo es la identificación de proteínas
inmunógenicas de BCG México.
Material y Métodos. Con un panel de 52 muestras séricas clasificadas en cuatro
grupos: pacientes con tuberculosis pulmonar activa (TB n=12), pacientes con
enfermedad pulmonar por micobacterias no tuberculosas, (MNTs n=11), sujetos
con reacción positiva al derivado proteico (PPD+ n=19) y sujetos con reacción
negativa al derivado proteico (PPD- n=10), se realizó la prueba de ELISA indirecta
para la titulación de IgG2 contra 16 cepas micobacterianas en tres grupos:
Complejo Mycobacterium tuberculosis (CMT n=4), M. bovis BCG (n=5) y MNT
(n=7). Los sueros con mayor título fueron utilizados para construir los
inmunoproteomas de las micobacterias. Para la primera dimensión se usaron tiras
de IPG (pH 4-7) y la segunda dimensión se corrió en geles de poliacrilamida SDSPAGE al 12.5% y fue transferido a membranas de PVDF.
Resultados. De cada grupo de sueros, se obtuvieron los títulos para IgG2 a partir
de los promedios de luminiscencia obtenidos por la técnica de ELISA indirecta
75
para cada una de las 16 cepas de micobacterias. Las medianas de los títulos para
los 52 sueros estudiados fueron de 56 con un IIC (47-78) y un IC 95% (20-510).
Los intervalos más amplios de títulos se encuentran en los sueros de pacientes
con tuberculosis pulmonar activa TB y en los sueros de pacientes con MNT. A
partir de la titulación de los sueros se seleccionó uno de cada uno de los cuatro
grupos, eligiendo aquellos que presentarán reactividad al mayor número de cepas
para su posterior uso en la inmunodetección a partir de Western blot para BCG
México. La inmunodetección de TB, MNT y PPD positivo y negativo contra las
proteínas de BCG México detectaron 18, 10, 20 y 3 proteínas respectivamente,
dando un total de 51 proteínas.
Conclusiones. El análisis comparativo entre las inmunodetecciones de TB, MNT,
PPD positivo y negativo para la cepa BCG México permitió la identificación de
proteínas únicas y compartidas. Las proteínas reconocidas por el suero de
paciente con TB pueden ser candidatas para la identificación de la enfermedad
pulmonar causada por CMT.
76
P14
MASS FINGERPRINTING OF Centruroides limpidus tecomanus (BUTHIDAE
SCORPION) VENOM AND BIOLOGICAL ACTIVITY OF VENOM FRACTIONS
L. L.Valdez-Velazquez1, L. J. G. Zamora- Pizano1, F. I. Coronas2, F.Z. Zamudio2,
C. Balderas2, L.D. Possani 2
1
Facultad de Ciencias Químicas, Universidad de Colima, Coquimatlán, Colima,
Mexico.2Instituto de Biotecnología, Universidad Nacional Autónoma de México,
Cuernavaca, Morelos, México. Email: [email protected]
Centruroides limpidus tecomanus is a Mexican scorpion of the state of Colima. It is
a medically important scorpion, but its venom is poorly studied. Here we report the
separation of the soluble venom of this scorpion by high-performance liquid
chromatography (HPLC). From 98 fractions obtained, 50 distinct components were
identified by electrospray mass spectrometry (LC/ESI-MS) analysis. The molecular
masses distribution of these components varies from 904 to 7555 Da, from which
48% falls in between 5500-7555 Da, 38% within the range of 3000-5500 Da and
12% within the range 900-3000 Da. The biological effects of the venom were
tested
on
crustacean/freshwater
shrimp
(Cambarellus
montezumae),
insects/cockroach (Acheta domestica) and mammals/mouse (strain CD1). For the
bioassays the HPLC fractions obtained during 60 minutes run were separated into
12 groups (5 min elution time each). Group VII was lethal to the three species
used for assay. The IV group had toxic effect in freshwater shrimps whereas
groups VI, VII and VIII were all lethal. For cockroach, groups V and VI were toxic
and group VII was lethal. In mouse the lethal components were found in group VII;
group VIII was toxic. The molecular masses in the mouse lethal group fall between
7013 and 7487 Da. Two peptides were obtained in homogeneous form. They were
lethal for the three kinds of animals used. They eluted from the HPLC at 32.72
minutes with molecular weight of 6334 Da and 31.85 min with 7430 Da,
respectively.
77
P15
EXTRACELLULAR PROTEIN-PROTEIN INTERACTIONS IN MIXED-SPECIES
BIOFILMS
Andrade-Domínguez A1., Trejo Hernández A., Meneses Moreno N., Romero
Martínez S.A., Encarnación Guevara S. M1.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México,
Programa de Genómica Funcional de Procariotes, Laboratorio de Proteómica.
C.P.62210. E-mail: [email protected]
Given the diversity of different cell types and the vast number of biological
processes that are in some way dependent upon cell surface interactions, it is
perhaps not surprising that a large proportion of eukaryotic genes may encode
proteins that are either secreted or have the potential to be exposed on the outer
surface of cells. Therefore, one of the greatest challenges of the post-genomic era
will be to define the full range of functions for each of the proposed extracellular
proteins. For secreted proteins, the goal will be to identify a comprehensive profile
of target receptors. However, in the case of transmembrane proteins, the challenge
is more complex. This class of proteins provides a vital means of communication
between extracellular and intracellular events. As such, these proteins participate
in a range of interactions with secreted proteins, proteins on the surface of other
cells, proteins within the same membrane, intracellular proteins and with proteins
from other species.
We propose a strategy based on Tandem Affinity Purification (TAP) in yeast for the
identification of extracellular interactions between proteins of yeast and bacteria,
which we call “mixed-interactomes”. We are investigating Saccharomyces
cerevisiae-R. etli and S. cerevisiae-Pseudomonas aeruginosa mixed-interactomes
in biofilms, with the aim of understanding the role of proteins secreted in the
interaction between microorganisms.
grant IN222707.
78
P16
PROLINE SUBSTITUTION REDUCES THE HEMOLYTIC ACTIVITY OF A
SHORT ANTIMICROBIAL PEPTIDE
Alexis Rodríguez1, Elizabeth Aguilar2, Elba Villegas2, Gerardo Corzo1
1
Cuernavaca, México, 2Centro de Investigación en Biotecnología, Universidad
Autónoma del Estado de Morelos, Cuernavaca, México. Email:
[email protected]
Melittin and Pardaxin are α-helical antimicrobial peptides highly hemolytic towards
red blood cells. The presence of a Proline residue at the midpoint regions of these
peptides introduces a rigid structural kink in their α-helix secondary structure. This
kink is associated to their high antimicrobial and hemolytic activities. On the other
hand, low hemolytic α-helical antimicrobial peptides such as Ponericins and
Oxypinins, have a more flexible structural kink represented by the motifs GPG and
GVG. Pandinin 2 (Pin2), from the venom of the scorpion P. imperator is an αhelical peptide with a Proline residue at the middle region of its structure that
resembles Melittin and Pardaxin. Pin2 also has antimicrobial activity in the
micromolar range against several bacteria, and a strong hemolytic activity. In this
work, the replacement of the proline residue in Pin2 by a GVG motif, as in
Oxypinins, was studied. Therefore, variants of Pin2 with residues V, GV, VG and
GVG instead of the proline were chemically synthesized. All Pin2 variants were
purified by HPLC, and their identities were verified using ESI mass spectrometry.
Circular dichroism measurements confirmed that all variants have α-helical
structures. All four Pin2 variants showed different antimicrobial activity against
Gram positive and negative bacteria in solid phase assays, being Pin2[GVG] the
most promising variant. However, in the liquid phase antimicrobial inhibition tested
against Staphylococcus aureus ATCC 25923, the MIC was not significant different
to all the variants (p>0.05). The hemolytic activity of the Pin2 variants from 0.16 to
25 µM was tested using human red blood cells, as result of the substitution of
proline, all variants showed lower hemolytic activity than the parental peptide,
being the peptide Pin2[GVG] the less hemolytic. Proline substitution by the motif
GVG improved the therapeutic index of Pin2.
Acknowledgements: This work was supported by grants from CONACyT
83962/2007 to E.V. and 49773/24968 to G.C. Alexis Rodriguez is beneficiary from
a Ph.D. scholarship from CONACyT.
Keywords: antimicrobial, peptide, arachnid
79
P17
IDENTIFICATION AND ANALISIS OF THE DIHYDRODIOL DEHYDROGENASE
(DDH) EXPRESION IN Aedes aegypti A MAJOR DENGUE VECTOR
Hernández Estrada M. G.1, García Gil de Muñoz F. L.2, Lánz Mendoza H.3,
Rodríguez MH.3, Hernández-Hernández F. C.1. 1CINVESTAV-IPN, Dpto.
Infectómica y Patogénesis Molecular, Lab. Entomología Molecular; 2Universidad
Simón Bolívar; 3CISEI-INSP, México. Email: [email protected]
Dihydrodiol dehydrogenases (DDHs) are enzymes catalyzing the NADP+ linked
oxidation of trans-di-hydrodiols of aromatic hydrocarbons rendering the
corresponding catechols. In mammals these enzymes participate in detoxification
of polycyclic aromatic hydrocarbons and inactivate circulating steroid hormones.
The best characterized DDH is the 3α-hydroxysteroid dehydrogenase/dihydrodiol
dehydrogenase (HSD/DD, 34-37kDa), monomeric or dimeric enzyme abundantly
expressed in kidney and rat liver that metabolize androgens, glucocorticoids and
prostaglandins (PGs). In invertebrates it has been showed that PGs regulate
cellular functions including immunity mechanisms but there are numerous
questions to investigate as presence of DDHs and its participation in PGs
metabolism. In this work, the presence of DDH in midguts of Aedes aegypti adult
females, a major Dengue vector, was demonstrated experimentally. The PGs
synthesis inhibitor Dexamethasone (Dex, a phospholipase A2 inhibitor) inhibited
protein synthesis and transcription of DDH in midgut and this effect was reversed
by PGs precursor arachidonic acid (AA). Beside this, DDH expression during
mosquito’s cycle of life is described. This is the first demonstration that DDH is
produced in mosquitoes and that its expression is regulated by Dex, a steroidal
anti-inflammatory agent. We plan to characterize the DDH biology in mosquito,
including its possible role on the regulation of immune response.
Key words: Aedes aegypti, Dihydrodiol dehydrogenase (DDH), 3α- Hydroxisteroid/
Dihydrodiol Dehydrogenase (3α HSD/DD), Dexamethasone (Dex).
80
P18
IDENTIFICATION OF DGKζ
ζ-SNX27 INTERACTION: NEW COMPONENTS OF
VESICULAR TRAFFICKING IN T LYMPHOCYTES.
Esther Rincón1, Teresa Santos-Mendoza1,2, Antonia Ávila-Flores1 and Isabel
Mérida1.
1
Department of Immunology and Oncology, National Center of Biotechnology,
Madrid, Spain. 2 National Institute of Respiratory Diseases, Mexico City, Mexico.
Diacylglycerol kinase ζ (DGKζ) is a member of the diacylglycerol kinase (DGK)
family of enzymes, which generate phosphatidic acid through diacylglycerol
phosphorylation. Previous reports demonstrated that DGKζ interaction with several
proteins is an important mechanism for the modulation of the localization and
activity of this enzyme. Here we used a proteomic approach to search for novel
DGKζ-interacting proteins and identified sorting nexin 27 (SNX27), a recently
described member of a protein family involved in intracellular trafficking. Coimmunoprecipitation and two-hybrid analysis confirmed PDZ-dependent
association between SNX27 and DGKζ. Since DGKζ is abundantly expressed in T
lymphocytes we characterized SNX27 expression and subcellular localization in
these cells. SNX27 co-localized with transferrin receptor (TfR)-positive vesicles,
suggesting a role for SNX27 in T cell endocytic recycling. Expression of deletion
mutants revealed that the SNX27 PDZ domain contributed to vesicle localization of
this protein, suggesting that interaction with DGKζ regulates SNX27 localization.
RNAi mediated knockdown of DGKζ showed accelerated TfR exit from the
lymphocyte endocytic recycling compartment back to the plasma membrane,
further confirming DGKζ-dependent control of vesicle trafficking. T lymphocyte
stimulation showed SNX27 polarization in endocytic/recycling endosomes to the
immunological synapse (IS). Altogether these data support a previously unreported
role for DGKζ in the modulation of membrane trafficking and suggest a function for
SNX27 at the IS interacting with PDZ-binding proteins.
81
P19
PROTEIN EXPRESSION PROFILE IN PLASMA OF OBESE CHILDREN
Esparza-Ibarra Montserrat1, Flores-Altamirano Fátima1, Pérez-Luque Elva1, GaraySevilla Ma. Eugenia1, Macías-Cervantes Maciste H1, Encarnación Sergio2 y
Pérez-Vázquez Victoriano1
1
División de Ciencias de la Salud. Departamento de Investigaciones Médicas.
Universidad de Guanajuato. Campus León. 2Centro de Ciencias Genómicas,
UNAM. E- mail: [email protected]
Obesity represents a serious threat to health of the population of almost every
country of the world. Recent data report an increment in the incidence and
prevalence in childhood obesity. It is associated with several risk factors for heart
and metabolic disease in adult life as hypertension, type 2 diabetes,
atherosclerosis and cancer. In obesity the organisms increment the white adipose
tissue which releases numerous proteins. We analyzed the protein expression
profiling in plasma of obese children and compare it to health children in order to
identify differentially expressed proteins. Eight children (8 to 9 yr-old) were
classified according body mass index in normal weight (n=4) and obese (n=4). We
measure glucose, triglycerides and cholesterol levels in blood samples. Proteins
were extracted of plasma and passed by affinity column to remove albumin and
IgG, bi-dimensional polyacrylamide gels (2D-PAGE) were performed and dyed with
Coomassie blue. Gels were digitalized and analyzed with Image Master 2D
Platinum software. Obese children showed high levels of triglycerides and low
levels of C-HDL. The 2D-PAGE analysis reports the presence of 22 spots in
samples of normal weight group and 24 spots in obese group. In 2D-PAGE we find
4 spots in obese children which were not present in health children and 2 spots
with less expression than health children. It will be important identified the proteins
with differential expression in order to find the possible functional relevance with
obesity. This work was support by Institutional Program 2008 of University of
Guanajuato.
82
P20
PROTEOMIC ANALYSIS OF MIXED BIOFILM Candida albicans Pseudomonas aeruginosa.
Trejo-Hernández A1., Andrade Domínguez A., Hernández Ortiz M., Encarnación
Guevara S.M.
Programa de Genómica Funcional de Procariotes, Laboratorio de Proteómica.
C.P.62210. E-mail: alison_82@ hotmail.com
The interactions among microbes inside the human body are of great importance
for the human health and the diseases development. For this reason the study of
interactions microbe - microbe is essential to understand microorganisms in vivo
activities such as commensal and pathogens. To know the molecular mechanisms
during the interaction C. albicans - P. aeruginosa, we analyzed the proteome of
both species when they form a mixed biofilm in conditions of hypoxia and
normoxia. With the aim to observe the differences in the proteomes of these two
microorganisms, we used 2D SDS-PAGE and masses spectrometry (MALDI-TOF)
for identifying differentially expressed proteins. From the proteome of P.
aeruginosa during mixed biofilm formation under hypoxia with C. albicans, where
we identified 156 proteins over 227 differentially expressed. Some of these
identified proteins are involved in the metabolism of Iron and in the biosynthesis of
the siderophore pyoverdine. Other proteins overexpressed in mixed biofilm under
hypoxia at 24 hrs. that we found are the following: proteases, external membrane
proteins, adherence and host invasion, also proteins involved in the metabolism of
fatty acids and unknown function. On the other hand the analysis of the proteome
of C. albicans when forms mixed biofilm with P. aeruginosa changes in ~118 spots.
Of these we identified proteins implied in glycolysis, drugs resistance and proteins
involved in host – pathogen interaction.
grant IN222707.
83
P21
EXTRACELLULAR PROTEOME OF Rhizobium etli IN EXPONENTIAL AND
STATIONARY GROWTH PHASE
Niurka Meneses Moreno1, Guillermo Mendoza Hernández2, Andrés Andrade
Domínguez1, and Sergio Encarnación Guevara1.
1
Genomics Sciences Center, Universidad Nacional Autonoma de Mexico,
Cuernavaca,México. 2Facultad de Medicina, Universidad Nacional Autónoma de
México, DF, México.
Studies of extracellular proteome have not been reported in Rhizobium etli. This
studies could discern many unknown questions about the extracellular proteins
functions, as well as the way they use to be exported; so our proposal was to
define the R. etli’s secretome and the role of the proteins that constitute it in the
cellular bacterium function. The proteins were identified by liquid chromatography
matched to the mass spectrometry. The proteins were grouped by COG (Cluster
Orthologous Groups) according to NCBI (National Center for Biotechnology
Information). In the exponential growth phase, 193 proteins were identified. It is
interesting that we found out, the related ribosomal proteins but we did not know
the role they could play when they are secreted. In the stationary growth phase,
191 proteins were identified; we found that the largest amount of identified proteins
for this growth phase, do not have an associated function and they represent
28.7% from the total of the identified proteins. We also analyzed the presence of a
peptide signal in the identified proteins, this allows us to suggest which of them
might have been secreting the by the secretion Type II. We demonstrate the
presence of vesicles of external membrane in R. etli. In the vesicles proteins and
other molecules are exported.
Part of this work was supported by CONACyT grant 60641 and DGAPA-PAPIIT
grant in 222707.
84
P22
LYT1 OLIGOMERIZE UNDER ACIDIC CONDITIONS: IMPLICATIONS IN THE
TRYPANOSOMA CRUZI INFECTION PROCESS
C. Lugo-Caballero1, G. Ballesteros-Rodea1, A. Rojo-Domínguez2, R. ManningCela1.
1
Molecular Biomedicine Department, CINVESTAV, Zacatenco, México.
2
Departamento de Ciencias Naturales y Departamento de Química, Universidad
Autónoma Metropolitana, Cuajimalpa, Unidad Iztapalapa, México
Few molecules have been identified that participate in the infection process of T.
cruzi but only LYT1 has been demonstrated by genetic analysis. Two LYT1
products are obtained by alternative trans-splicing in which the full-length LYT1
protein contains an amino-terminal signal sequence and an internal sequence for
directs nuclear localization, whereas the truncated protein lacks the secretion
sequence. Therefore, one form of the LYT1 protein is secreted and participates in
hemolysis, infectivity and the parasitoforous vacuole escape whereas the other
form is located in the kinetoflagellar zone and is involved in the parasite
developmental process. This dual/single-gene expression, and consequent
differential localization and functional switching of protein products expose these
molecules to different microenvironments that could impact on protein folding and
interaction with other proteins. In this work we obtained the LYT1 interactome and
evaluated the LYT1-LYT1 interaction using in vitro and in vivo approaches. The
results show that LYT1 interacts with itself and with at least other 14 proteins from
8 to 255 kDa. The MS-MS analysis allows us to identify proteins that are related
with the infection or stage-transition process. Besides, we also demonstrated a
direct full-length LYT1-LYT1 multimeric interaction, possibly through a coiled-coil
region included between the 163 to 198 protein residues. The sequence analysis of
this membrane insertion domain related LYT1 with lytic pore forming proteins. This
is the first work where it has been determined the interaction map of a T. cruzi
protein.
85
P23
RHIZOBIUM ETLI PHOSPHOPROTEOME DURING FREE LIFE AND
SIMBIOSYS METABOLISM.
Magdalena Hernández-Ortiz, Angel Gabriel Martínez-Batallar, Sandra ContrerasMartínez, Agustín Reyes-Pérez, Yolanda Mora, and Sergio Encarnación.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México.
Protein phosphorylation is the most studied posttranslational modification (PTM),
which reversibly regulates of most cellular process. Although phosphorylation
cascades are typical for eukaryotes also prokaryotes proteins are phosphorylated,
in previous works has been established proteins phosphorylation in bacteria.
Rhizobium etli is a soil bacteria that has two life stiles: free life and symbiosis whit
Phaseolus vulgaris, both are highly regulated. In preliminary studies we found a
special pattern of phosphorylated proteins. Currently we are using metal oxide
affinity chromatography (MOAC) to enrich phosphoproteins and analyzed whit twodimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectrometer during free life
and symbiosis metabolism. We could identify more abundant proteins, which are
different in 2D gel before enrichment. Searches within Netphos server
(http://www.cbs.dtu.dk/services/NetPhos/)
and
FindMod
(http://www.expasy.ch/tools/ findmod/) predicted that most of identified proteins
had at least one Phosphorylation site. More than, in most of cases we founded the
mass peptide phosphorylated and non phosphorylated. We will show some
identities of these proteins.
grant IN222707
86
P24
PROTEOMICS ANALYSIS IN BIOFILM FORMATION IN Rhizobium etli CE3: A
SCREENING OF DIFFERENT STAGES
Reyes-Pérez Agustín1,2, Hernández Ortiz Magdalena, Martínez-Batallar Ángel
Gabriel, Aguilar-Vera Alejandro, Vargas-Lagunas María del Carmen and
Encarnación Guevara Sergio 1.
1
Cuernavaca, México, 2 Doctorado en Ciencias Biológicas, Facultad de Ciencias,
Universidad Nacional Autónoma de México, México D.F. Email:
[email protected]
Most microbes as Rhizobium etli, grow in the rhizosphere as organised biofilm
communities on surfaces, Sessile bacteria (biofilm), constitute a major component
of the bacterial biomass in many environments. Bacterial cells attached to, and
growing on surfaces in mature biofilms are physiologically distinct from their
planktonic counterparts. We are analysing with proteomics methodology the
phenotypic changes that take place when planktonic cells of R. etli CE3 make the
transition to the biofilm mode of growth.
Results
Our preliminary results indicate that the biofilm proteome differed from the
planktonic (sessile) proteome. The sessile proteome, at early stages (24 and 72
hours), differed from the more developed biofilm (240 days) proteome. In addition,
using confocal microscopy we observed structural differences in the biofilm at
different stages of develoment shown structural differences, mainly between early
stages against late stages. We conclude than the observed difference was not due
to a single factor; rather, it was due to a multitude of up- and down-regulated
proteins, and posttranslational modification of proteins may also have been
involved in the process of biofilm formation.
Part from this work is supported by CONACYT grant 60641 and DGAPA IN222707 grant.
87
P25
ANALYSIS OF PROTEIN PROFILES OF ANTIBIOTIC-RESISTANT Salmonella
Typhimurium
Lugo-Melchor OY1, Barba de la Rosa AP2, Fagersquist CK3, Leon-Felix J1 and
Chaidez C1
1
Centro de Investigación en Alimentación y Desarrollo, Culiacan, Sinaloa, México,
2
Instituto Potosino de Investigacion Cientifica y Tecnologica, San Luis Potosi,
Mexico, 3Western Regional Research Center, United States Department of
Agriculture, California, USA. Email: [email protected]
The emergence of resistant strains to antibiotics, such as the multi-drug resistant
Salmonella Typhimurium is of particular global concern due to its frequent isolation.
Beside there is a lack of information about the kind of proteins expressed by
antibiotic-resistant S. Typhimurium strains so it will provide a basis for developing
an intervention. In this study, 2D-PAGE and mass spectrometry were utilized to
obtain protein profiles of two environmental isolates of multidrug-resistant S.
Typhimurium, tetracycline-resistant S. Typhimurium and an antibiotic-sensible S.
Typhimurium. Total protein extracts were prepared for each isolate by an acid
extraction method and analyzed by 2D-PAGE. Proteins profiles were compared
among them and differential proteins were identified by mass spectrometry (LCMS/MS). The obtained data was compared using MASCOT, NCBI and Swiss-Prot.
Similar pattern was observed among them. Many of the identified proteins in the
proteome of all isolates included mainly cytosolic proteins, outer membrane
proteins (OMPC), protein biosynthesis (elongation factor Ts, chaperone protein
and ribosomal proteins) and metabolic proteins. It was noted that flagellin phase 1
protein was absent in sensible S. Typhimurium but present in multidrug-resistant S.
Typhimurium. On the other hand, tetracycline-resistant S. Typhimurium showed a
flagellin isoform. These results demonstrated that global proteins are related to
bacterial survival and flagellin phase 1 could be involved in virulence of antibioticresistance in comparison with sensible Salmonella. Proteomic analysis was found
to be a useful tool for identifying specific proteins expressed in antibiotic-resistant
and sensible S. Typhimurium.
88
P26
IDENTIFICATION OF ANTIGENS EXPRESSED DIFFERENTIALLY BETWEEN
ISOLATES OF HIGH AND LOW PREVALENCE OF MYCOBACTERIUM
TUBERCULOSIS.
Vargas-Romero F.1, Hernández-Pando R.2, Mendoza-Hernández G.3, GutiérrezNájera N.4 & Castañón-Arreola M.1
1
Genomic Sciences Program, UACM,México2 Experimental Pathology, INCMNSZ,
México,3Medical School ,UNAM, México 4 Proteomic, INMEGEN, México.
[email protected]
Tuberculosis is one of three mainly infectious diseases worldwide. Still there are
not well defined all the factors involved in the virulence of the strains, but there is
thought that there are related to Mycobacterium tuberculosis (M.tb) invasion
capacity and to the expression of specific genes related to their survival, immune
evasion and growth.
Strains of Beijing genotype family have a high impact on the current TB worldwide
epidemics, mainly on endemic countries like Cape Town, South Africa. To identify
culture filtrate proteins differentially expressed between the hypervirulent isolate
Sud31 and hypovirulent isolate sud23, we use 2D-PAGE and DIGE. Selected
proteins were identified by MALDI. Killing assay was used to evaluate differences
in bacterial resistant to elimination by macrophages. We identify five proteins
differentially expressed in the isolate sud31 (CPF10, Ag85B, TRX, TF and
hypothetical protein) which can be involved in virulence of isolate. We also
observed that sud31 isolate is two folds more resistant to degradation inside
macrophages than Sud23 isolate.
89
P27
OPTIMIZATION OF A PROTEIN EXTRACTION METHOD SUITABLE FOR A
FERMENTED MAIZE DOUGH PROTEOMIC STUDY
Cárdenas M. C.1, Delgado L1., Wacher C.2, Barkla B.3 and Rodríguez-Sanoja R.1
1
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de
México, México D. F., 2Facultad de Química, Universidad Nacional Autónoma de
México, México D. F. 3Instituto de Biotecnología, Universidad Nacional Autónoma
de México, México D. F. Email: [email protected]
Fermented maize dough (pozol) contains low amounts of protein but high amount
of starch. Current methods for vegetal protein extraction cannot produce high
quality samples for proteomic studies. The high starch concentration and its
inherent gelatinization ability cause several problems. To deal with these
inconveniences we employed different methodologies for isolation, purification, and
solubilization of proteins. We obtained a reproducible methodology for high quality
pozol protein extraction. These extracted proteins were solubilized and examined
using 1-D and 2-D electrophoresis. The results showed the need to employ a
sequential combination of extraction approaches for increasing proteome
coverage.
90
P28
SHOTGUN PROTEOMICS AS A STRATEGY FOR THE CHARACTERIZATION
OF WATER STRESS PROTEIN-RESPONSIVENESS IN AMARANTH
(Amaranthus hypochondriacus L.)
Bischof Sylvaina, Roschitzki Berndb, Reiland Sonjaa, Baginsky Sachaa, Barba de la
Rosa Ana Paulinaa,c
a
Plant Biotechnology, ETH-Zurich, bFunctional Genomics Center Zurich, University
of Zurich, cMolecular Biology Division, Institute for Scientific and Technological
Research in San Luis Potosi, IPICYT. E-mail: [email protected];
[email protected]
Proteomics has attracted great attention in systems biology because it generates
knowledge about the concentrations, interactions of proteins, which are the major
structural and functional determinants of cells. The term “shotgun proteomics” is
used to describe mass spectrometry-based methods that enable the rapid
identification of proteolytic peptides from complex protein mixtures in a datadependent manner.
Abiotic stress is the main factor negatively affecting crop growth and productivity
worldwide. Efforts are directed towards a better understanding of the genetic basis
of the adaptive response of plants to drought and salinity to exploit this knowledge
for breeding purpose. Amaranth is a plant tolerant to salinity and drought and
produces seeds of high nutritive and nutraceutical properties. For these reasons,
amaranth is proposed as a non-model plant for studying the phenomenon of
tolerance to abiotic factors.
The objective of this work was to apply shotgun proteomics for the analysis of
amaranth leaf proteins and identify proteins (genes) that are differentially
expressed when the plant is grown under water deficit.
Amaranthus hypochondriacus var. Nutrisol was grown in a growth chamber and 4weeks seedlings were subjected to water stress. After one week of stress, the
fourth leaf was collected and frozen immediately at -80°C. The leaves were milled
to get a fine powder and total protein was extracted. Proteins were separated in a
12% SDS-PAGE, the gel was divided in 15 parts and proteins were digested “in
gel”. Peptides were extracted, and analyzed in an LTQ-FT (Thermo). MS and
MS/MS data were analyzed with the Mascot and SEQUEST search algorithms.
The data revealed the differential regulation of most of the enzymes responsible for
photosynthesis and proteins related to abiotic stress tolerance.
AP Barba thanks to ETH-Zürich and Sabbatical Fellowship-CONACyT.
91
P29
SEPARATION OF CALCIUM-DEPENDENT PROTEIN KINASES FROM
BEETROOT PLASMA MEMBRANES ACCORDING TO THEIR HYDROPATHY,
AND THEIR IDENTIFICATION BY MASS SPECTROMETRY
M.B, Lino Alfaro1, A. Chagolla López2, F.C. Alcántar Aguirre1 L.E. González de la
Vara1
1
Departamento de Biotecnología y Bioquímica, Cinvestav-IPN Unidad Irapuato,
Irapuato, México, 2Cinvestav-IPN Unidad Irapuato, Irapuato, México. Email:
[email protected]
Many plant plasma membrane proteins are involved in signal transduction, Ca2+dependent protein kinases (CDPK) among them. These enzymes phosphorylate
many plasma membrane transport proteins, including the H+-transporting ATPase,
in response to cytoplasmic Ca2+ signals. In beetroot plasma membranes, we have
found that most of the protein kinase activity is Ca2+-dependent (1), and that the
H+-ATPase is inhibited by a Ca2+-dependent phosphorylation (2). In order to
separate membrane proteins according to their hydropathy, we have developed a
method based in serial phase-partitioning with the detergent Triton X-114 (3).
Using this method, and determining the sizes of Ca2+-dependent kinases in “in-gel”
assays, at least five different kinases with different hydropathies were found. Of
these, three kinases were purified further and identified by MS/MS: 1) a 60-kDa
kinase that, depending on the beetroots’ growth conditions, can be hydrophobic,
amphipathic or hydrophilic. This kinase was identified as a CDPK similar to rice
OsCDPK2 (OsCPK19) and to arabidopsis AtCPK9. It probably forms complexes,
since its determined sedimentation coefficient was 12S. 2) A 50-kDa hydrophilic
kinase, identified as a CDPK very similar to the 60-kDa one (4); and 3) a 75-kDa
hydrophobic kinase similar to STRUBBELIG-receptor family receptor-like kinases
from several plant species.
(1) Baizabal-Aguirre VM, González de la Vara LE (1994) Physiol Plant 91:147-54.
(2) Lino B, et al. (1998) Planta 204:352-59. (3) González de la Vara LE, Lino B.
(2009) Anal Biochem 387: 280-86. (4) Lino B, et al. (2006) Planta 225:255-68.
92
P30
LOSS OF STARCH BIOSYNTHESIS ENZYMES DURING THEIR EXTRACTION
AND PURIFICATION ANALYZED BY ELECTROPHORESIS
Juárez-García, E., Agama-Acevedo, E. and Pacheco-Vargas, G.
Centro de Desarrollo de Productos Bióticos del IPN, Apartado postal 24 C.P.,
62731, Yautepec, Morelos, México. Phone: +54 739 42020, Fax: +52 739 41896,
e-mail: [email protected]
Starch biosynthesis enzymes can be located in the surface and/or associate (in the
inner) to granule. The methods to separate these enzymes involve using aqueous
buffers, provoking that some starch synthases are lost during starch washing step,
principally those surface-associated proteins, while granule-associated protein are
not affected. The objective of this work was to evaluate the loss of starch
synthases during their extraction and purification by electrophoretic analysis. Corn
from self-pollinnated ears were collected 20 days after pollination (dap). Starch
biosynthetic enzymes from corn endosperm were separated using extraction and
SDS buffers. After each wash with the buffer the sample was centrifugated and
supernatant was collected. To extract granule-associated enzyme (GA), the pellet
was dissolved with SDS-buffer and the mixture was gelatinized (boiling water) for
15 min, cooling down, centrifugated and the supernatant was collected. Each
supernatant was mixed with cold acetone-TCA to precipitate proteins, which were
analyzed by electrophoresis. One-dimensional electrophoresis showed several
proteins bands in the extraction and SDS buffers with molecular weight (MW)
approximately of 59, 75 and 86 kDa, which have been reported for granule bound
starch synthase (GBSS), starch synthase (SS) and starch branching enzyme
(SBEIIb), respectively. Predominant protein of approximately 60 kDa and diffuse
band of 257 kDa were found in GA samples. Two-dimensional electrophoresis of
proteins separated with extraction and SDS buffers presented several spots with a
pI between 4.5-8.5 and pI 4.2-5.6, respectively, and MW 59-86 kDa which indicates
they were isoforms of starch synthases. Extraction buffer used to eliminate storage
proteins from corn endosperm also separated starch synthase presented in the
surface and granule-associate (GBSS). This GBSS can be eliminated from the
inner of the granule because the starch granules in the endosperm at 20 dap does
not have compact crystalline matrix. The washing process with aqueous buffers
containing a detergent and reducing agent can separate surface and granuleassociated proteins, even though the starch has not been gelatinized.
93
P31
ANALYSIS OF THE RESPONSE OF Musa sp. Var. "Yangambi Km5" LEAVES
TO INFECTION CAUSED BY Mycosphaerella fijiensis Morelet USING
PROTEOMIC TECHNIQUES
L. Escobar-Tovar1, M. Guzmán2, J. Sandoval2, M. Gómez-Lim1
1
CINVESTAV-IPN, Irapuato, Guanajuato, México
2
CORBANA, Guápiles, Costa Rica
Bananas and plantains (Musa spp.) are the fourth major crop worldwide after rice,
wheat and maize constituting the staple food for millions of people in tropical
countries. However, the crop is severely affected by Mycosphaerella fijiensis
Morelet, the fungus that causes black Sigatoka disease, which is characterized by
necrosis of leaf tissue, poor yield and premature ripening of the fruit. Although
many genes coding for resistance proteins have been identified in banana, the
molecular mechanisms underlying the disease are unknown. There are some wellknown resistant varieties to the fungus such as Yangambi Km5 (AAA, subgroup
Ibota), but the resistance has been broken by the fungus in some countries such
as Costa Rica. Since this isn’t the case of Mexico’s crops, it was of our interest to
compare the response of Yangambi to both strains using proteomic techniques.
We hope to identify differential proteins that may get induced/repressed during the
infection of Yangambi with both fungical strains. In theory, the same proteins
should present a contrasting expression pattern when the Yangambi is infected
with the less virulent strain from Mexico. In any case, the results should give us
some information about the molecular mechanisms underlying the plant response
to the disease. The methodology for plant infection of Yangambi under controlled
conditions, for protein extraction from leaves and for the electrophoresis running
conditions in the first dimension had been established. The next step is to identify
proteins differentially expressed between Yangambi infected with one fungal strain
or the other in 2-D gels. Once identified, they will be sequenced and a search
performed in databases for possible annotation.
94
P32
COMPARATIVE PROTEOMICS OF CHLOROPLAST BIOGENESIS AFFECTED
Arabidopsis thaliana MUTANTS
A. Arturo Guevara-García1, Gabriel Martínez Batallar2, Maricela Ramos-Vega1,
Odette Avendaño Vázquez1, Carolina San Román1, Patricia León1 & Sergio
Encarnación Guevara2
1
Cuernavaca, México, 2Centro de Ciencias Genómicas, Universidad Nacional
Autónoma de México, Cuernavaca, México. Email: [email protected]
The life on earth depends on photosynthesis, a process that occurs principally in a
singular organelle of the plant cells named chloroplast. In fact, chloroplasts are
responsible for a several essential plant functions, including CO2 fixation and
biosynthesis of fatty acids, pigments and amino acids. Chloroplast biogenesis
alludes to the conversion of proplastids (small non differentiate organelles lack of
inner membranes which are presents in meristematic plant cells), into chloroplasts
through a complicated process, modulated by environmental and developmental
signals, that requires the coordinate expression of nuclear- and chloroplastencoded genes to culminate with a functionally mature chloroplast. For several
years, our research has been focusing over the chloroplast biogenesis. Thus, using
biochemical, genetic and molecular strategies, we had identified some genes
involved in that interesting differentiation program. Our achievements include a
PPR protein involved in the chloroplast RNA-processing and enzymes from
chloroplast MEP and carotene pigment pathways. Now, with the idea of identify
novel chloroplast biogenesis genes, we are interested in a comparative proteomic
approach. Our study includes the comparison of the 2D-PAGE protein profiles
from three pigment-affected mutants (cla1, clb5 and clb19), with the subsequent
identification of some of the differentially expressed proteins by mass
spectrometry. The advances about the analysis of the 2D-PAGE protein profiles
will be discussed during the meeting.
95
P33
ANALYSIS OF PAPAYA RIPENING-ASSOCIATED PROTEINS: A PROTEOMIC
APPROACH
Huerta-Ocampo José Ángel1, Barrera-Pacheco Alberto1, Lino-López Gisela2,
Osuna-Castro Juan Alberto2, Barba de la Rosa Ana Paulina1.
1
Instituto Potosino de Investigación Científica y Tecnológica A.C. San Luis Potosí,
México. 2Facultad de Ciencias Biológicas y Agropecuarias, Universidad de
Colima.Colima, México. Email: [email protected]
Papaya (Carica papaya) is a climacteric fruit susceptible to postharvest losses due
to the fast softening caused by ethylene. 1-MCP (1-methylcyclopropene) an
inhibitor of ethylene action and innocuous to human health has proved to extend
the postharvest life in papaya ‘Maradol’ fruits as many as 50% when treated with
300 ppb of 1-MCP. When quality attributes were determined, color and firmness
losses were significantly retarded, while the loss of weight, pH and total soluble
solids were not significantly affected. To detect biochemical changes the protein
expression pattern in response to 1-MCP treatment versus the control condition
were analyzed by two-dimensional electrophoresis (2-DE). Unripe fruits were
treated with 300 ppb 1-MCP, control and treated samples were collected after 3, 6,
9, 12, 15 and 18 days after the 1-MCP application. The fruits were peeled, seeds
removed and the pulp was cut into small pieces, frozen with liquid nitrogen and
finely powdered with an electric grinder and stored at –80°C until its use. Protein
extraction was carried out by a phenol-based protocol followed by trichloroacetic
acid precipitation. Tandem mass spectrometry identification of differentially
expressed proteins among the 2-DE gels will allow us to elucidate the mechanisms
and proteins involved in the papaya ripening process.
This work was supported by FOMIX-Colima-2008-C01-81701
96
P34
PAPAYA FRUIT β-GALACTOSIDASE: A TWO-DIMENSIONAL GEL
ELECTROPHORESIS STUDY
Lino-López Gisela Jareth1, Barba de la Rosa Ana Paulina2, Osuna-Castro Juan
Alberto1
1
Laboratorio de Biotecnología de la Facultad de Ciencias Biológicas y
Agropecuarias, Universidad de Colima, Tecomán, Colima México. 2Instituto
Potosino de Investigación Científica y Tecnológica A.C. San Luis Potosí, México.
E-mail: [email protected]
Papaya is a commercially relevant fruit for Mexico, but has a short postharvest life
due to its rapid softening during the ripening. The β-galactosidase (β-Gal) is a
glicosyl hydrolase that reduces the levels of cell wall galactosyl residues [β-(1→4)galactan bonds] in fruits as tomate, kiwifruit and papaya, contributing to cell wall
modification and their softening. In papaya fruit the application of 1methylcyclopropene (1-MCP), an inhibitor of the ethylene functions, increased the
firmness and declined the β-Gal activity. The non-1-MCP-treated fruits showed a
significant reduction in the firmness and rise in the β-Gal activity with galactose
loss from cell wall pectin, suggesting that the β-Gal is an important enzyme in the
papaya postharvest softening process. Total proteins of papaya cv. maradol ripe
fruit were extracted with sodium citrate buffer, pH 4.6, and the β-Gal purified with
an ion-exchange chromatography (CM-Sepharose). The β-Gal self-assembly ability
was studied by native polyacrylamide gel electrophoresis (PAGE) and twodimensional PAGE (2-DE-PAGE), and its corresponding Western blot. Also the βGal was analyzed in denaturing 2-DE-PAGE. This investigation will help to
understand the structure-function relationship of the β-Gal in postharvest softening
of papaya fruit. This work was supported by grants CONACyT-FOMIX-COLIMA
and FRABA-Universidad de Colima.
97
P35
TWO-DIMENSIONAL GEL ELECTROPHORESIS-BASED DISULFIDE BOND
ANALYSIS OF AMARANTH 11S GLOBULIN EXPRESSED IN Escherichia coli
BY A THIOL-SPECIFIC PROBE
Cuellar-Olalde Rocio1, Barba de la Rosa Ana Paulina2, Osuna-Castro Juan
Alberto1.
1
Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima. Colima,
México.
2
Instituto Potosino de Investigación Científica y Tecnológica A. C. San Luis Potosí,
México. Email: [email protected]
The 11S globulin is one of the most important amaranth seed storage proteins with
a good balance of essential amino acids, food functional and nutraceutical
properties. The hexameric amaranth 11S globulin has an apparent molecular
weight of 300-400 kDa comprising 53-kDa subunits, each of which consists of a
34-kDa acidic polypeptide and a 22-kDa basic polypeptide linked by an interchain
disulfide bridge, and other intrachain disulfide bond in the acidic polypeptide.
Accumulating evidence suggests that reduction-oxidation (redox) regulation play
important roles in a broad spectrum of biological processes as seed germination by
thioredoxins and appropriate folding of disulfide bond-rich proteins.
The amaranth 11S globulin expressed in E. coli was purified to homogeneity by
anion-exchange and size-exclusion chomatographies. The pure 11S globulin was
disolved in the SDS-sample buffer with and without 5 mM DTT and incubated at
room temperature and different reduction times. Then, monobromobimane (mBBr),
a fluorescent thiol-specific probe, was added to a final concentration of 1 mM and
the solution was stored for 15 min. To identify the 11S globulin disulfide bonds, the
amaranth recombinant protein was analyzed by 2-DE gels using immobilized linear
pH gradient strips (pH 4-7). The resultant gels were examined under 365-nm UV
light to detect mBBr-labeled disulfide bridge. The analysis of disulfide bonds and
redox changes in the E. coli recombinant 11S globulin would allow for understand
the mechanism of its in vitro and in vivo folding.
98
P36
Localization study of internal or surface proteins in starch granule from
mature maize endosperm
R.G. Utrilla-Coello1, S. L. Rodríguez-Ambriz1, A. P. Barba de la Rosa2, H. LanzMendoza3, G. Hurtado-Sil3 & L.A. Bello-Pérez1
1
Centro de Desarrollo de Productos Bióticos,Instituto Politécnico Nacional
2
Instituto Potosino de Investigación Científica y Tecnológica, 3Instituto Nacional de
Salud Pública. Email: [email protected]
In recent years, our knowledge of the enzymes involved in starch biosynthesis
have greatly increased because they could help understanding the molecular
structure of starch. In total thirteen enzymes are involved in the starch
biosynthesis, but only three enzymes are considered as key points during the
process: starch synthase (SS), starch branching enzyme (SBE) and starch
debranching enzyme (SDBE). This work was carried out a proteomic study of
internal and surface proteins of starch granule isolated from white and blue maize
endosperms. Starch biosynthesis enzymes were extracted. Sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE) were used to separate and identify
the starch biosynthesis enzymes. Matrix-assisted laser desorption/ionization time
of flight mass spectrometry (MALDI-TOF/MS) analysis was carried out to identify
spots of proteins from the SDS-PAGE and 2D-PAGE. The electrophoretic patterns
were different for both maize starches. SDS-PAGE showed that several (more
definition on the proteins will be better) proteins with different molecular
weightshave been eliminated during the extraction and washing process to obtain
starch granule associated proteins (SGAPs), where the main proteins in this
fraction are starch soluble synthase II (SSSII) and granule bound starch synthase
(GBSS) isoform I. In the eliminated protein groups, three isoforms of SBE (two in
blue maize endosperm gel (SBEI and SBEIIa) and one in white endosperm gel
(SBEIIb)) were found, which indicates that SBE existed in the periphery granule or
in the soluble fractions. Additionally, other enzymes, as acetyl-CoA carboxylase,
sorbitol dehydrogenase, calcium-dependent protein kinase, were found but not
related with starch biosynthesis, The results from 2D-PAGE in the concentrated
extract of SGAPs showed that GBSSI was present in both samples with an
isoelectric point range between 5 and 6 with 7 spots. This indicates that GBSSI is a
multi-enzymatic complex that depends on the extent of GBSSI participating in the
amylose biosynthesis. The three enzymes (SSSII, GBSS and SBE) related to
starch biosynthesis were found due to: a) the isolation procedure used; b) enzyme
location (surface or internal) in starch granule and c) mature maize endosperm.
99
P37
MODERN TECHNIQUES FOR PURIFICATION AND
IDENTIFICATION OF PROTEINS: A REVIEW
Márquez-Ipiña, A. R.1, Ascacio-Martínez, J.A.I1., Acuña-Askar, K.2, BarreraSaldaña, H.A. 1
1
Departamento de Bioquímica y Medicina Molecular, 2 Laboratorio de
Biorremediación Ambiental, Departamento de Microbiología, Facultad de Medicina,
Universidad Autónoma de Nuevo León Monterrey, N.L., México
Molecular functionality and purity are major issues when recovering recombinant
proteins from production processes. A review on protein characterization
processes reveals that recent strategies have been developed towards the use of
metal-coded affinity tags (MeCAT) and isotope-coded affinity tags (ICAT) as
molecular techniques for protein purification and quantification purposes. The
appropriate use of proteins for clinical applications on patient clinical treatment
protocols or environmental bioprocesses requires high degree of certainty as to the
structural integrity, including adequate folding to hold proper functions. Various
analytic techniques, including one and two-dimension electrophoresis, LC-MS
electrospray, LC-MS/MS and MS-MALDI-TOF-TOF have been used to monitor
substitutions or losses in aminoacid sequences, oxidations, methylations,
deamidations or any other significant modifications on protein structure. Since
metabolic functioning relies on protein expression, the quality of clinical treatments
or environmental bioremediation processes can be anticipated by monitoring
proteome expression. In conclusion, recently developed affinity tag molecular
techniques along with modern analytic techniques offer the opportunity to bring
forth high quality evidence of the steps involved in production processes of
recombinant proteins.
100
P38
IMPLEMENTATION OF A MALDI-IMAGING PLATFORM IN RAT LIVER TISSUE
Calderón-González K.G.*1, Regalado-Santiago D.J.*1, 2, López-Luna A.2, Monroy
Núñez G. Y.1,3 Quintanar-Jurado V.1, Pérez-Carreón J. I.1, Gimeno M.2, Bárzana
E.2, Villanueva-González P.2, Gallegos-Pérez J. L.1
1
Instituto Nacional de Medicina Genómica, México, D.F., 2 Facultad de
Química,Universidad Nacional Autónoma de México, México, D.F., 3Facultad de
Química, Universidad Autónoma de Baja California. Email:
[email protected], [email protected]
* Both authors contributed equally to this work
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry
(IMS) is a powerful tool for investigating the distribution of proteins and small
molecules within biological systems through the in situ analysis of tissue sections.
Hundreds of peptides and proteins can be simultaneous mapped in a tissue with a
lateral resolution of approximately 30–50 µm. Matrix is first uniformly deposited
over the surface of the tissue section, utilizing procedures optimized to minimize
protein migration. Proteins are then desorbed from discrete spots or pixels upon
irradiation of the sample in an ordered array or raster of the surface. Each pixel
thus is keyed to a full mass spectrum consisting of signals from protonated species
of molecules desorbed from that tissue region. A plot of the intensity of any one
signal produces a map of the relative amount of that compound over the entire
imaged surface. The objective of this work was to implement MALDI-IMS and
optimize the matrix application on rat liver tissue in order to obtain better MSsignals. Matrix was deposited by sublimation in high vacuum. For this, a
homemade flat-bottom condenser was designed to which glass coverslips or
MALDI plate inserts could be fixed. Rat liver samples (10 µm thickness) were
covered using different matrices: 2,5-dihydroxybenzoic acid (DHB), α-cyano-4hydroxycinnamic acid (α-CHCA), and 3,5-dimethoxy-4-hydroxycinnamic acid
(sinapinic acid). Results showed that sublimation matrix deposition offered a good
particle distribution on the tissue and therefore better MS-signals and images.
Keywords: MALDI imaging, Mass spectrometry, Matrix deposition.
101
P39
IN DEPTH, COMPREHENSIVE MAPPING OF THE HUMAN SEMINAL
PLASMA PROTEOME BY A NOVEL, ITERATIVE LC-MS/MS/DATABASE
SEARCH WORKFLOW
Claire Dauly1, Antonie D. Rolland1, Martin Hornshaw2, Regis Lavigne3, and
Charles Pineau2,3
1
Thermo Fisher Scientific, 16 Avenue du Québec, 91963 Courtaboeuf cedex,
France; 2TInsem U625, Campus de Beaulieu, 35042 Rennes cedex, France; 3
Proteomics Core Facility OUEST-GENOPOLE, Inserm U625, Campus de
Beaulieu, 35042 rennes cedex, France
The purpose is maximize sequence coverage and protein identification for the
analysis of non-liquefied human seminal plasma proteome using iterative LCMS/MS and exclusion lists.
Methods: A list of peptides which were confidently identified from the first LCMS analyses was generated with Proteome Discoverer software and used as a
dynamic exclusion list during a second analyses of the sample. Peptides
identified from the first and the second acquisition were further excluded during
a third analyses of the sample.
Results: This strategy allowed the identification of additional peptides which
were not characterized in the first LC-MS/MS analyses. Low copy number
proteins were identified and overall protein coverage was increased, leading to
more than 800 proteins confidentially identified (5% FDR).
102
P40
INTACT PROTEIN SEQUENCING USING ETD AND PTR IN A DUALPRESSURE LINEAR ION TRAP
Zhiqi Hao1, Jae C. Shwartz1 and Andreas FR Huhmer1
1
Thermo Fisher Scientific, San Jose, CA,USA
To evaluate the performance of proton transfer reaction (PTR) in a linear ion
trap for charge reduction following electron transfer dissociation (ETD) and to
investigate the utility of ETD with PTR for intact protein sequencing.
Electron transfer dissociation (ETD) is an beneficial tool for intact protein
analysis because it is relatively insensible to the size, amino acid composition
and posttranslational modifications of proteins, therefore randomly cleaves
protein / peptide backbone bonds. ETD of intact proteins performs with high
efficiency, generating very informative, yet extremely complex spectra which
contain highly charged product ions that are difficult, or even impossible to
resolve at unit resolution. Proton transfer reaction (PTR) following ETD was
developed to reduce spectral complexity. PTR removes protons from the
multiply charged product ions, generating a simplified spectrum that contains
product ions of resolved charge states at unit resolution. PTR has recently
been implemented, under software control, in the LTQ XL ion trap and the new
LTQ Velos dual-pressure linear ion trap. Shown below is the instrument
configuration of LTQ Velos ion trap with ETD and PTR. The new technologies
in LTQ Velos instrument not only improve the sensitivity by 5-10 times, but also
improve trapping, isolation and dissociation efficiencies. The dual-pressure trap
provides two times faster overall scan speed compared to LTQ XL instrument.
Here, the performance of ETD PTR for intact protein analysis is compared
between the LTQ XL and LTQ Velos instruments.
Methods: Standard peptides were purchased from Anaspec. Intact proteins
were purchased from Sigma. Desalted intact protein was diluted in acetonitrile /
water / formic acid (50:50:0.1) to a final concentration of 1 to 5 pmol/µl. The
sample was directly infused using static nanospray with a 4 micron tip
(PicotipTM, New Objective). ETD with PTR was performed using Thermo
Scientific LTQ XL ETD and LTQ Velos ETD mass spectrometers with PTR
capability under LTQ 2.6 instrument control software with developer’s kit.
Benzoic acid anions which are generated in the chemical ionization source at
the rear of the instrument were used as PTR reagent. The anion target was
2e5. Activation time was 5 - 10 msec for ETD or for 25 -50 msec for PTR.
103
Results: When intact protein ETD analysis is performed on a unit-resolution
instrument,the resulting spectra are information rich, yet contain multiply
charged product ions which can not be resolved adequately. PTR following
ETD reduces charge carried by product ions so that they are resolved at unit
resolution. Using different PTR time, ETD-PTR of intact proteins generates
very informative and well-resolved spectra. The improved sensitivity,
resolution, and faster scan rate of the LTQ Velos mass spectrometer resulted
in extensive sequence coverage of intact proteins of up to 30KDa. With product
ions of +5, +6 and even +7 charge resolved in full, zoom scan mode, the LTQ
Velos instrument identified many c’/z. product ions as big as 10 kDa in the
complex spectrum of intact myoglobin and carbonic anhydrase in a single 10
minute experiment. Number of identified ions, thus the sequence coverage was
significantly improved in LTQ Velos compared to LTQ XL.
104
P41
QUANTITATIVE PROTEOMIC APPROACHES FOR THE DETERMINATION
OF SERUM PROTEOME IN PATIENTS WITH BENIGN PROSTATE
HYPERPLASIA AND PROSTATE CANCER
Theodoros Roumeliotis1, Nicolae Eugen Damoc2, Michaela Scigelova2, Martin
Hornshaw2, Eugenia G. Giannopoulou3 ,Thomas Moehring2and Spiros D.
Garbis1
2
Thermo Fisher Scientific, Bremen, Germany; 1Biomedical Research
Foundation of the Academy of Athens, Greece; University of Peloponnese,
Tripoli, Greece.
Purpose: The development of a novel quantitative MS method for the
characterization of the low molecular weight (< 50 kDa) serum proteome of
potential clinical significance to prostate cancer.
Methods: An LTQ Orbitrap Velos mass spectrometer equipped with
progressively stacked-ring ion guide, dual-cell differentially pumped ion trap,
and higher energy collision dissociation (HCD) cell with axial field was used for
the analysis of the iTRAQTM 8-plex labeled sample set.
Results: Using ZIC-HILIC pre-separation, a broad coverage of human serum
proteome was achieved with respect to protein MW, pI, hydrophobicity and
biological significance. The LTQ Orbitrap Velos instrument delivered both
identification and quantitation of peptides in a single method/analysis. Our
findings from serum analysis support the notion of epidemiological correlation
of metabolic syndrome to prostate cancer proteins found in prostate tissue.
Uniquely occurring prostate cancer (PCa) proteins were identified relative to
the benign prostate hyperplasia (BPH).
References:
1. Garbis, S.D., Tyritzis, S.I., Roumeliotis, T., et al. Search for potential markers for prostate
cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific
isobaric tagging (iTRAQ) with two dimensional liquid chromatography and tandem mass
spectrometry. (2008) Journal of Proteome Research, 7 (8), pp. 3146-3158.
2. Josefsson, A., Wikström, P., Granfors, T., et al. Tumor size, vascular density and
proliferation as prognostic markers in GS 6 and GS 7 prostate tumors in patients with long
follow-up and non-curative treatment. (2005) European Urology, 48(4): 577-83.
3. Nakajima, T, Kubota, N., Tsutsumi, T. et al. Eicosapentaenoic acid inhibits voltage-gated
sodium channels and invasiveness in prostate cancer cells.(2009) British Journal of
Pharmacology 156(3):420-31. 4. Kouri, E.D., Labrou, N.E., Garbis, S.D., et al. Molecular and
Biochemical characterization of the Parvulin-type PPIases in Lotus japonicus. (2009) Plant
Physiology PMID:19403733.
105
P42
PROTEOMIC ANALYSIS OF ACTIN STRUCTURES FORMED AFTER
TROPHOZOITES-FIBRONECTIN (FN) AND TROPHOZITES-HOST
INTERACTION
Javier Reyna R. 1, Hernández Ramírez V.I.1, Guzmán Bautista E.R.1, Cázares
F.1, Galván I.2, Gallegos Pérez J.L.3, Calderón González K.G.3, TalamásRohana P.1
1
Departmento de Infectómica y Patogénesis Molecular y 2Laboratorios
Centrales; CINVESTAV-IPN; 3INMEGEN, México, D.F. México.
[email protected]
Entamoeba histolytica causes amoebic dysentery and liver abscess. In
response to FN, trophozoites form stress fibers, phagocytic invaginations,
adhesion plates, and actin spots. The aim of this work was to identify
components of actin stress fibers and other actin structures. Trophozoites were
adhered to uncoated or FN-coated substrates. After trophozoites disruption
with lysis buffer, stress fibers were extracted with detergent. Silver staining
analysis of proteins obtained from stress fibers in both conditions was done in
10 % SDS-PAGE, finding proteins from 30 to 200 kDa molecular weight range.
Actin and and ROCK-2 were detected by Western blot. Further proteomic
analysis by MALDI TOF/TOF of stress fibers from trophozoites incubated with
FN allowed the identification of actin, myosin heavy chain, Rab family GTPase,
and Arp2/3 complex, in contrast with trophozoites incubated on glass;
furthermore, proteomic analysis of the cytoskeleton fraction from trophozoites
recovered from hepatic lesions, showed that calreticuline is present during
actin cytoskeleton structuration at the host-parasite interphase. These results
confirm the participation of structural and regulatory proteins during the
formation, in E. histolytica, of actin cytoskeleton rearrangements.
106
P43
ANALYSIS BY 2D-ELECTROPHORESIS AS A PREELIMINARY STEP FOR
FURTHER MASS SPECTROMETRY ANALYSIS OF ASCITIC FLUID SAMPLES
FROM OVARIAN CANCER PATIENTS
Garibay Cerdenares O.L.1, Osorio C.1, Morales Vásques F.2, Gallardo Rincón D.2,
Talamás Rohana P.1
1
Centro de Investigación y de Estudios Avanzados, I.P.N., México D.F.
2
Instituto Nacional de Cancerología, México D.F.
Ovarian Cancer is the fifth most common in women worldwide. The abdominal
distension is a symptom that occurs with gradual accumulation of ascitic fluid in the
peritoneal cavity. Tumoral cells are able to survive as a single cell or as aggregates
in the ascites. To date there are very few methods for ovarian cancer diagnosis.
Detection in serum of cancerous CA 125 antigen has been proposed, however its
specificity is low and its elevation has been reported in various disorders; therefore
there is a critical need for generating new diagnostic methods. The aim of this
project was to define a methodological strategy to process corporal fluid samples
for further processing by Mass Spectrometry after sample processing that included
protein enrichment using alcohol-acetone precipitation followed by a protocol to
deplete albumin before sample analysis by double dimension polyacrylamide gels.
Eight ascites samples from ovarian cancer patients classified to be in clinical
phases III and IV have been processed. Results show that the procedure followed
to clean the samples is simple and reproducible and allows good resolution in 2Dgels.
107
P44
ANÁLISIS PROTEÓMICO COMPARATIVO DE TUMORES ONDONTOGÉNICOS
HUMANOS
García-Muñoz A.1, Cázares-Raga FE1., Hernández-Hernández FC1., BolognaMolina R2., Rodriguez M.A1.
1
Depto. de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, México, D.F.
Facultad de Medicina, Universidad de Guadalajara, Guadalajara, Jalisco Hospital
Juárez de México. email: [email protected]
2
En la cavidad bucal la mayoría de los tumores son benignos, sin embargo éstos
suelen ser localmente agresivos y clínicamente tratados con hemimandibulectomía
o la resección en bloque de los maxilares. Los tumores odontogénicos derivan de
los tejidos formadores de dientes y pueden ser de estirpe mesenquimal o epitelial.
Hasta ahora se conoce muy poco de los mecanismos, moléculas y factores
desencadenantes de las lesiones tumorales orales. El objetivo de este trabajo es
estudiar los perfiles proteómicos de diferentes tumores odontogénicos. Los
tumores analizados fueron el mixoma, de estirpe mesenquimal, usando como
control al folículo dental (FD) y varios subtipos de lesiones de estirpe epitelial
como el ameloblastoma sólido multiquístico (ASM), ameloblastoma uniquístico
(AU), carcinoma ameloblastico (CA) y quiste dentígero (QD, control). El análisis
de las proteínas se realizó por electroforesis en una y dos dimensiones. Los
resultados mostraron que el mixoma y el folículo dental, comparten muchas
proteínas con algunas diferenciales. Mientras que al comparar, el ASM, AU, CA y
un QD que son lesiones tumorales de origen epitelial, se observaron diferentes
patrones proteicos, aun cuando las lesiones se conforman por el mismo tipo de
células pero con diferencias clínicas entre los tumores. Una vez concluido el
análisis de los patrones protéicos con el programa Image Master 2D Platinum, las
proteínas diferenciales serán identificadas por espectrometría de masas y sus
niveles de expresión serán estudiados en los diferentes tipos de tumores.
Palabras clave: tumores odontogénicos, mixoma, ameloblastoma, proteómica.
108
P45
A PROTEOMIC ANALYSIS OF THE SECUNDARY EVENTS ASSOCIATED
WITH THE APPLICATION OF SCORPION TOXIN Cn2 ON EXCITABLE CELLS
Pedraza Escalona, MM1, Restano-Cassulini, R1., Batista, C.V.F1., Possani LD1.
Cuernavaca, México.
Email:[email protected]
Scorpionism is a public health problem in Mexico, with a registered number of
accidents in excess of 260,000 per year. The primary events of intoxication are
well known and documented. They are due to the presence of short chain peptides
(toxins) that recognize ion-channels on excitable cells and modify their normal
function, either by blocking the ion-conductance (K+-channels) or altering the
gating mechanism of the channel (Na+-channels) (Rodríguez & Possani, Toxicon
46 831-844]. However, the secondary events following the binding to the primary
target of the toxins is poorly understood. Here we describe the secondary events
caused by application of Cn2, the mayor β-toxin from the venom of the scorpion
Centruroides noxius, on F11 cells in culture. This cell line comes from a dorsal root
ganglion neuroblastoma that expresses different ion channels. Proteomic analysis
by two-dimensional gel electrophoresis followed by mass spectrometry identified
44 proteins whose expression levels changed after different times of exposition to
the toxin: 26 were down-regulated and 18 were up-regulated. Bioinformatic
analysis revealed differential expression of cytoskeletal components, molecular
chaperones, enzimes, DNA-binding proteins and components of RNA-processing
pathways.
Acknowledgements: Supported in part by SEP_CONACyT grant # 4864. 6 to
LDP
109
ÍNDICE DE AUTORES
A
Acosta-Pérez
Acuña-Askar
Agis-Juárez
Agama Acevedo
Aguado-Santacruz
Aguilar
Aguilar-Vera
Aillet
Alcántar Aguirre
Alcaráz Flores
Alvarez
Alvarez-Sánchez
Amador-Gaytán
Andrade Domínguez
Angel-Ortiz
Arroyo
Ascacio-Martínez
Avendaño-Vázquez
Ávila-Flores
Avila-González
M
K
R
E
A
E
A
F
FC
S
S
ME
E
A
C
R
JAI
O
A
L
L38
P37
L08
P30
L16
P16
P24
L13
P29
L08
L11
P04, P07
P13
P15, P20, P21
L33
L33, L42, P05, P07
P37
P32
P18
L33, L42
Baginsky
Balderas
Ballesteros-Rodea
Barba de la Rosa
S
C
G
AP
Barkla
Barrera-Pacheco
Barrera-Saldaña
Bárzana
Batista
Bautista-de Lucio
Bello Pérez
Benitez
Bernhardt
Bhoumik
BJ
A
HA
E
CVF
VM
LA
O
J
A
L35, P28
P14
P22
L35, L37, L41, P25, P28,
P33, P34, P35, P36
L15, P27
P33
P37
P38
L03, L38, P11, P12, P45
L32
P36
L12
L23
L04
57
100
19
93
28
79
87
25
92
19
23
66, 69
75
78,83,84
52
52,61,67,69
100
95
81
52,61
B
54,91
77
85
54,56,60,88,91
96,9798,99
27,90
96
100
101
14,57,73,74,109
51
99
24
38
15
110
Bischof
Bologna-Molina
Borbolla-Vázquez
S
R
J
L35, P28
P44
P09
54,91
108
71
K
R
MC
RE
M
AI
R
FE
F
R
A
C
S
A
H
S
RN
S
FI
L
G
JL
A
R
L07, L31, P06, P08, P38,P42
L02, L05
P27
L33
P07, P26
P13
P11, P12
L29, P09, P10,P44
P42
L06
L16 , L17, P29
P25
L08
L09
L06
L11
L06
L25, L34, L40, P23
P14
L29, P09
P16
L31
L36
P35
S
NE
C
A
RM
L
C
L07
P41
P39
L37
L08
P27
L31
18
105
102
56
19
90
50
M
L34
53
C
Calderón-González
Caprioli
Cárdenas
Cárdenas-Guerra
Castañón-Arreola
Castillo-Rodal
Castro Franco
Cázares-Raga
Cázarez
Chaerkady
Chagolla López
Chaidez
Chavez-Salinas
Checa Rojas
Chen
Chen
Cole
Contreras Martínez
Coronas
Cortéz-Martínez
Corzo
Cruz-Colín
Cruz-Hernández
Cuellar-Olalde
18,50,68,70,101,106
13,16
90
52
69,89
75
73,74
48,71,72,108
106
17
28,30,92
88
19
20
17
23
17
42,53,59,86
77
48,71
79
50
55
98
D
Damían-Zamacona
Damoc
Dauley
De León-Rodríguez
del Angel
Delgado
Díaz-Tufinio
E
Elizalde
111
Easterling
Elortza
Encarnación Guevara
ML
PF
S
England
Escobar-Tovar
Esparza-Ibarra
Espitia-Pinzón
L
M
CI
L20
L13
L09, L25, L30, L34, L40, P03, P15,
P19,P20, P21, P23, P24, P32,P46
L13
P31
P19
L26
CK
SB
F
EC
MA
P25
L04
L30 ,P19
L30
P04
D
JL
I
ME
SD
FL
A
Y
OL
JA
B
EG
C
M
J
M
LE
M
M
A
AA
GJ
T
LH
N
M
P43
L07, L31, P06, P08, P38, P42
P42
P19
P41
P09, P17
P44
L32
P43
P10
L35
P41
L41
P38
L11
P31
L17, P29
L26
L06
L16
P32
L04
P08
P01
L31, P08, P26
P31
34
25
20,42,49,53,59,65,78
82,83,84,86,87,95,110
25
94
82
44
F
Fagersquist
Ficarro
Flores-Altamirano
Flores-Pérez
Fonseca-Sánchez
88
15
49,82
49
66
G
Gallardo-Rincón
Gallegos-Pérez
Galván
Garay-Sevilla
Garbis
García Gil de Muñoz
García-Muñoz
Garfias
Garibay-Cerdenares
Garibay-García
Gerrits
Giannopolou
Gil
Gimeno
Goldsmith
Gómez-Lim
González de la Vara
González -Zamorano
Gucek
Guerrero-Rangel
Guevara-García
Gutierrez
Gutiérrez-Espíndola
Gutiérrez-González
Gutiérrez-Nájera
Guzmán
107
18,50,68,70,101,106
106
82
105
71,80
108
51
107
72
54
105
60
101
23
94
30,92
44
17
28
95
15
70
63
50,70,89
94
112
Guzmán Bautista
ER
H
Hao
Z
Hardesty
W
Hernández
M
Hernández G
R
Hernández Orihuela
L
Hernández Pando
R
Hernández-Coronado M
Hernández-Estrada
MG
Hernández-Hernández FC
Hernández-Ortiz
M
Hernández-Ramírez
VI
Herrera
Y
Herrera-Carrillo
Z
Herrera-Salgado
Y
Higareda
V
Higareda Almaraz
JC
Hjerpe
R
Hornshaw
M
Hoving
S
Huerta-Ocampo
JA
Huhmer
AFR
Hurtado-Sil
G
P42
P40
L05
L09, L25, L34
L39
P11
P26
L15
P17
L29, P09, P10, P44
P03, P20, P23, P24
P42
L25
P02
L34, L40
L12
L09, P03
L13
P39, P41
L23
L37, P33
P40
L38, P36
106
103
16
20,42,53
58
73
89
27
80
48,71,72,108
65,83,86,87
106
42
64
53,59
24
20,65
25
102,105
38
56,96
103
57,99
I, J, K
Izquierdo
Javier Reyna
Jianjung
Jiménez Corona
Juárez Garcia
Kaspar
Kim
J
R
H
A
E
S
MS
L12
P42
L06
L07
P30
L01
L06
V
J
H
R
P
J
L13
P01
L12, L38, P17,P36
P39
P32
P25
24
106
17
18
93
12
17
L
Lang
Langridge
Lánz-Mendoza
Lavigne
León
Leon-Felix
25
63
24,57,80,99
102
95
88
113
Lino-Alfaro
Lino-López
Lloret-Sánchez
López Luna
López-Briones
López-Camarillo
López-Espinosa
López-Vidal
Lopitz-Otsoa
Lugo-Caballero
Lugo-Melchor
Luna
Luviano-Bazán
MB
GJ
L
A
S
C
NL
Y
F
C
OY
JP
D
L17, P29
P33, P34
P13
P38
L30
P04
L32
L24, P13
L13
P22
P25
P04
L38
MH
E
R
D
G
AR
AG
J
AG
F
JL
J
A
T
G
G
N
EP
I
K
HP
T
GY
Y
F
A
P19
P08
P22
L30
L06
P37
L34
L38
L25, P23, P24, P32
L40
L41
L07
L01
P01
L39
L25, L26, L32, L37, P04, P21, P26
L25, P15, P21
P11
P18
L06
L01,L18
P41
P06, P38
L25, P23
P43
L07
30,92
96,97
75
101
49
66
51
41,75
25
85
88
66
57
M
Macias-Cervantes
Maldonado Olvera
Manning-Cela
Mares-Álvarez
Marjan
Márquez-Ipiña
Martínez
Martínez-Barnetche
Martínez-Batallar
Martínez-Obregón
Martínez-Salgado
Mas Oliva
Matros
McKenna
Mendoza
Mendoza Hernández
Meneses Moreno
Menezes
Mérida
Min-Sik
Mock
Moehring
Monroy Nuñez
Mora
Morales-Vásques
Moreno
82
70
85
49
17
100
53
57
42,86,87,95
59
60
18
12
63
58
42,44,51,56,66,84,89
42,78,84
73
81
17
12,31
105
68,101
42,86
107
18
114
O, P, Q
Ortega-López
Ortiz-Plata
Osorio
Osuna-Castro
Pacheco Vargas
Pandey
Pando-Robles
Pantoja
Paredes-López
Pascual
Pedraza-Escalona
Pérez Carreón
Pérez-Luque
Pérez-Vázquez
Peters
Pimienta
Pineau
Pitarch
Possani
Prieto Conaway
Quintanar Jurado
Quintas Granados
J
A
C
JA
G
A
V
O
O
N
MM
JI
E
V
EC
G
C
A
LD
MC
V
LI
L33
L29
P43
P33, P34, P35
P30
L06
L12
L15
L36
L04
P45
P38
P19
L30, P19,
L04
L04, L06
P39
L41
P14,P45
L19,L21
P38
P07
52
48
107
96,97,98
93
17
24
27
55
15
109
101
82
49,82
15
15,17
102
60
77, 109
32,35
101
69
P
AC
J
E
LA
M
DT
S
FJ
O
R
J
JP
A
R
E
C
L39
L09
L30
P02
L33, L42, P05
P32
P38
L35, P28
L33, L42, P05
L25
P45
L08
L07, P06
P23, P24
P42
P18
L06
58
20
49
64
52,61,67
95
101
54,91
52,61,67
42
109
19
18,68
86,87
106
81
17
R
Ramírez R
Ramírez Torres
Ramírez-Emiliano
Ramon-Gallegos
Ramón-Luing
Ramos-Vega
Regalado Santiago
Reiland
Rendón Gandarilla
Resendis
Restano-Cassulini
Reyes del Valle
Reyes-Grajeda
Reyez-Perez
Reyna
Rincón
Robert
115
Rodríguez
Rodríguez
Rodríguez
Rodríguez
Rodríguez Ambriz
Rodriguez Sanoja
Rodríguez-Cuevas
Rojo-Domínguez
Rolland
Romero Martínez
Ronai
Rosas-Cárdenas
Roschitzki
Roumeliotis
A
MA
MS
MH
SL
R
S
A
AD
SA
Z
FF
B
T
P16
P44
L13
P09, P10
P36
P27
P04
P22
P39
P15
L04
L36
L35, P28
P41
79
108
25
71,72
99
90
66
85
102
78
15
55
54,91
105
C
H
J
T
S
M
U
VV
JC
P
C
J
JM
S
P32
L29, P09
P31
P18
L06
P41
L01
L38
P40
P10
L14
L23
L11
L06
95
48,71
94
81
17
105
12
57
103
72
26
38
23
17
P
LM
M
A
RG
P42,P43
L22, P01
L13
P15, P20
P36
106,107
37,63
25
78,83
99
SE
LL
J
L16
P14
L23
S
San Román
Sánchez-Hernández
Sandoval
Santos-Mendoza
Saraswati
Scigelova
Seiffert
Serrano-Pinto
Shwartz
Sierra-Martínez
Silva-Sánchez
Stegemann
Strul
Sukumar
T, U
Talamás-Rohana
Terán
Torres-Ramos
Trejo-Hernández
Utrilla Coello
V
Valdés-Rodríguez
Valdez-Velázquez
van Oostrum
28
77
38
116
Vargas Lagunas
Vargas-Romero
Vázquez-Carrillo
Ventzki
Vera-Estrella
Villa-Hernández
Villanueva González
Villegas
Vissers
MC
F
LI
R
R
O
P
E
H
L25, P24
P26
P07
L23
L15
P11, P12
P38
P16
L27
42,87
89
69
38
27
73,74
101
79
45
C
JT
D
W
K
W
S
M
G
LJG
FZ
E
M
P27
L10, L28
L01
L01
L01
L26
L06
L08
106
P14
P14
L32
L11
90
22,46
12
12
12
44
17
19
17
77
77
51
23
W,X Y, Z
Wacher
Watson
Weier
Weschke
Witzel
Xolalpa
Xu
Yocupicio Monroy
Yien
Zamora-Pizano
Zamudio
Zenteno
Zhu
117

III Simposio Mexicano de Espectrometría de Masas 1

Transcription

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