III Simposio Mexicano de Espectrometría de Masassmp.org.mx/nsmp/files/Memorias simposio2009.pdf · III Simposio Mexicano de Espectrometría de Masas 8 Programa y Resúmenes III Simposio

III Simposio Mexicano de Espectrometría de Masas

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III Simposio Mexicano de

Espectrometría de MasasEspectrometría de MasasEspectrometría de MasasEspectrometría de Masas

Proteómica Celular y MolecularProteómica Celular y MolecularProteómica Celular y MolecularProteómica Celular y Molecular

Ciudad de San Luis Potosí

8 al 12 de Noviembre 2009


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iner.salud.gob.mx

ipicyt.edu.mx

cinvestav.mx


iner.salud.gob.mx inmegen.gob.mx

ipicyt.edu.mx

cinvestav.mx

ccg.unam.mx


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inmegen.gob.mx

ibt.unam.mx

insp.mx

biofound.org


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Programa y Resúmenes

III Simposio MexicanIII Simposio MexicanIII Simposio MexicanIII Simposio Mexicano deo deo deo de

Espectrometría de MasasEspectrometría de MasasEspectrometría de MasasEspectrometría de Masas

Proteómica Celular y MolecularProteómica Celular y MolecularProteómica Celular y MolecularProteómica 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 6 2

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, UN AM

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 HER2- positive 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


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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, and secretome in Rhiobium etli.

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


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RESÚMENES

CONFERENCIAS PLENARIAS

Salón Real San Luis “A”


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L01

EVALUATION OF PLANT GENETIC RESOURCES USING PROTEOM IC APPROACHES

KATJA WITZEL1, STEPHANIE KASPAR1, DIANA WEIER1, WINNIE WESCHKE1, UDO

SEIFFERT2, ANDREA MATROS

1 & HANS-PETER MOCK1

1Leibniz 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 PROCES SES

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.


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L03

PROTEOMICS ANALYSIS OF AMPHIBIAN SKIN SECRETIONS US ING 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, 2Pascual J.

1 Current Address: Department of Molecular Biophysics and Biochemistry, Yale

University New Haven, CT. USA

2Previous 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.


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L06

PROTEOMIC IDENTIFICATION OF NOVEL BIOMARKERS FOR HE R2- POSITIVE BREAST CANCER

Pimienta Genaroф1

, Yien Gu2, Xu Sun

3, Jianjun Hu

3, Kim Min-Sik

1, Chaerkady

Raghothama1, Gucek Marjan

4, Cole Robert N

4, Sukumar Saraswati

5, Pandey

Akhilesh1,6

and Chen Hexin2¶

1 McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry,

Johns Hopkins School of Medicine, Baltimore, Maryland, USA 2Department 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 5Sidney 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.


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L07

PROTEOMIC ANALYSIS OF OXIDATIVE STRESS IN A DIET-IN DUCED HYPERCHOLESTEROLEMIC MODEL

J. P. Reyes-Grajeda 1, 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.


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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, DEN-2, 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 post-traduccionalmente, 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.


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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 disulfide-isomerase, Vinculin, Ezrin, 14-3-3 protein zeta/delta, Heat shock 70 kDa protein 1 and 78 kDa glucose-regulated protein.


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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 CaM-kinase 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 SDS-PAGE 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.


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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.


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L11

PROTEOMICS OF PLANT REDOX REGULATORY NETWORKS

Sixue Chen 1*, Mengmeng Zhu1, Johanna M. Strul2, Sophie Alvarez1, Justin Goldsmith1

1Department of Biology, University of Florida, Gainesville, FL 32610, USA

2Life 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.


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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.


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L13

FUNCTIONAL CHARACTERISATION OF UBIQUITYLATED PROTEI N COMPLEXES USING TANDEM UBIQUITIN BINDING ENTITIES ( TUBEs):

TOWARDS THE UBIQUITIN INTERACTOME

M.S. Rodriguez 1, R. Hjerpe1, F. Lopitz-Otsoa 1, F. Aillet1, M. Torres-Ramos1, V. Lang1, P. F. Elortza2, England3.

1Proteomics 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.


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L14

FLUJO DE TRABAJO PARA DESCUBRIMIENTO DE BIOMARCADOR ES 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.


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L15

QUANTITATIVE PROTEOMICS OF TONOPLAST FROM THE HALOP HYTE MESEMBRYANTHEMUM CRYSTALLINUM REVEALS NOVEL ASSOCIATIONS AND FUNCTIONS OF GLYCOLYTIC ENZYMES IN PLANT SALT T OLERANCE

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 (2D-DIGE) 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 co-ordinated 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.


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L16

PROTEOMIC ANALYSIS OF THE RESPONSE TO OSMOTIC STRES S IN BOUTELOUA GRACILIS

1 S.E. Valdés Rodríguez ., 1 A. Guerrero Rangel., 2 A. Aguado Santacruz, 1 A Chagolla López.

1Departamento 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


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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).


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L17

SEPARATION OF BEETROOT PLASMA MEMBRANE PROTEINS ACC ORDING TO THEIR HYDROPATHY AND THEIR IDENTIFICATION BY MAS S

SPECTROMETRY

L.E. González de la Vara 1, B. Lino Alfaro1, A. Chagolla López2

1Departamento 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.


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L18

CHARACTERIZATION OF PLANT CO-CHPAERONES WITH HOMOLO GY 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

.


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L19

ADVANTAGES OF HIGH MASS ACCURACY, RESOLVING POWER A ND 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


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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.


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L20

TOP-DOWN PROTEOMICS, A MULTI-PLATFORM PRIMER

Michael L. Easterling 1

1Bruker 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.


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L21

A NOVEL DUAL PRESSURE ION TRAP FOR HIGH THROUGHPUT PROTEIN IDENTIFICATION AND HIGHLY SENSITIVE ANALYSI S 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.


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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.


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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.


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L23

3D-GEL ELECTROPHORESIS, A NEW APPROACH TO PROTEIN A NALYSIS

Robert Ventzki 1, Sjouke Hoving2, Jörg Bernhardt1,3, Jan van Oostrum4, and Josef

Stegemann1

1Ernst-Moritz-Arndt University, Institute for Microbiology, 17487 Greifswald, Germany

2Novartis Institutes for BioMedical Research, Developmental & Molecular Pathways, 4002 Basel, Switzerland

3DECODON 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 laser-induced 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.


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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 SDS-PAGE 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


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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, laser-induced 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.


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L24

IDENTIFICACIÓN Y ANÁLISIS COMPARATIVOS DE LOS PROTE OMAS DE

MICOBACTERIAS NO TUBERCULOSAS DE AISLAMIENTOS CLÍNI CO 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.


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PROTEOME, PHOSPHOPROTEOME AND SECRETOME IN Rhizobium etli

1Sergio Encarnación, 1Magdalena Hernández, 1Gabriel Martínez-Batallar, 1Osbaldo Resendis, 1Sandra Contreras, 1Niurka Meneses, 2Guillermo Mendoza,

1Yesenia Herrera, 1María del Carmen Vargas and 1Yolanda Mora

1Centro 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


43

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.


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HOST-PATHOGEN INTERACTIONS IN TUBERCULOSIS: A PROTE OMIC APPROACH

1Clara Inés Espitia Pinzón ,2Gillermo Mendoza-Hernández, 1Wendy Xolalpa and

1Margarita González-Zamorano

1Departamento 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.


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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.


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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.


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SESIONES ORALES

Salón Real San Luis “A”

15:30 – 16:30


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L29

PROTEOMIC ANALISYS OF EFFECT PRODUCED BY FOCAL CERE BRAL ISCHEMIA IN A RAT MODEL

Sánchez-Hernández H .1, Cázares-Raga FE.1, Cortés-Martínez L.1, Hernández-Hernández FC.1, Ortiz-Plata A.2

1Depto. de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, Av. IPN 2508, Col. Sn Pedro Zacatenco, México, D.F. 07360, Tel. 57473800 Ext. 5646, E;

2Instituto 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 MALDI-TOF/MS and cell processes activated in response to injure will be suggested.

Key words: Cerebral ischemia, sanguineous reperfusion, differentially expressed proteins


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L30

DIFFERENTIAL PROTEOMIC ANALYSIS IN LIVER OF DIABETI C DB/DB MICE

Flores-Pérez Elsa C 1, Mares-Álvarez Daniela P1, Flores-Altamirano Fátima1, Ramírez-Emiliano Joel1, López-Briones Sergio1, Encarnación Sergio2 y Pérez-

Vázquez Victoriano1

1Departamento 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.


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L31

POTENTIAL BIOMARKER IN BREAST CANCER DERIVED FROM P ROTEOMIC ANALYSIS: BIOCHEMICAL CARACTERIZATION OF PYRUVATE-K INASE 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. E-mail: [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.


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L32

DIFFERENTIAL PROTEIN EXPRESSION IN HUMAN PTERYGIUM

Bautista-de Lucio VM 1, 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 stress-induced 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.


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L33

AN IMMUNOPROTEOMIC APPROACH TO IDENTIFY POTENTIAL BIOMARKERS FOR Trichomonas vaginalis

L.A. Ramón-Luing1, F.J. Rendón-Gandarilla1, R.E. Cárdenas-Guerra2, J. Ortega-López2, L. Avila-González1, C. Angel-Ortiz1, and R. Arroyo 1

1Departamento 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.


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L34

ENRICHMENT OF PHOSPHOPEPTIDES BY METAL ORGANIC AFFI NITY 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. Email: [email protected]

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.

Part of this work was supported by CONACYT grant 60641 and DGAPA-PAPIIT grant IN222707


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L35

PHOSPHOPEPTIDE ANALYSIS IN Arabidopsis thaliana SUBJECTED TO HIGH LIGHT GROWTH CONDITIONS

Barba de la Rosa AP ab, Reiland Sonjaa, Bishof Sylvaina, Roschitzki Berndc, Gerrits Bertranc, Baginsky Sachaa

aInstitute for Scientific and Technological Research in San Luis Potosi, México. bPlant 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 16-h 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 C- and 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.


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L36

PROTEOMIC OF PRICKLY PEARS WITH CONTRASTING RIPENIN G BEHAVIOR

Rosas-Cárdenas F.F .1, Paredes-López O.1 , Cruz-Hernández A.1

1Depto 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.


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L37

PROTEOMIC ANALYSIS OF AMARANTH UNDER DROUGHT STRESS

Huerta-Ocampo José Ángel 1, Mendoza-Hernández Guillermo2, De León-Rodríguez Antonio1, Barba de la Rosa Ana Paulina1

1Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí, México.

2Facultad 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. Two-dimensional 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 down-regulation 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


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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-Mendoza 2.

1Centro 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.


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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 TroA-Like 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.


59

L40

PHOSPHOPROTEOME ANALYSIS OF THE S accharomyces cerevisiae IN THE ANSWER TO OXIDATIVE STRESS.

Martinez Obregon F. 1, 2, Herrera Salgado Y.2, Contreras S.2 and Encarnación Guevara S.2

1The 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 wild-type 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.


60

L41

TWO-DIMENSIONAL BLUE NATIVE/SDS GEL ELECTROPHORESIS OF Candida albicans MULTIPROTEIN COMPLEXES

J. Luis Martínez Salgado 1, Aida Pitarch2, Ana Paulina Barba de la Rosa1 and

Concha Gil2 1Instituto Potosino de Investigación Científica y Tecnológica, San Luis Potosí,

México.

2Departamento 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 first-dimension 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/SDS-PAGE 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.


61

L42

IDENTIFICATION OF THE SUBPROTEOME OF Trichomonas vaginalis ACTIVE ENDOPEPTIDASES: THE DIFFERENTIAL EXPRESSION OF PROT EINASES

BY IRON OR CELL CONTACT

L.A. Ramón-Luing , F.J. Rendón-Gandarilla, L. Ávila-González, and R. Arroyo

1Departamento 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-D-zymograms, 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 200-10 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, TvCP4-like, 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.


62

RESÚMENES DE PRESENTACIONES EN CARTEL

Salón Real San Luis “B”


63

P01

PROTEOMIC ANALYSIS OF NASAL SECRETIONS FROM CHILDRE N SUFFERING PARAINFLUENZA VIRUS INFECTION

L. H. Gutiérrez-González1, J. Langridge2, T. McKenna2, L. M. Terán1

1Instituto Nacional de Enfermedades Respiratorias, México, D. F., México, 2Waters, 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.


64

P02

PROTEIN BIOMARKERS USEFUL IN A DIFFERENTIAL SKIN CA NCER 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 formalin-fixed 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, biomarker, skin cancer, LC-MS, bidimentional electrophoresis Preference: POSTER PRESENTATION


65

P03

ANALYSIS OF THE GLOBAL PATTERNS OF PROTEIN EXPRESSI ON IN CELLULAR LINES OF CERVICAL UTERINE CANCER

Juan Carlos Higareda Almaraz1, Magdalena Hernandez Ortiz1, Sergio Encarnación 1

1Centro 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.


66

P04

PROTEOMIC ANALYSIS IDENTIFIED GLIOXALASE I AS A OVE REXPRESSED 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*

1Universidad Autónoma de la Ciudad de México, Posgrado en Ciencias

Genómicas, México DF. 2Instituto de Enfermedades de la Mama, FUCAM. 3CINVESTAV, 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 LC-MS/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.


67

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.


68

P06

EVALUATION OF PARAFFIN-EMBEDDED TISSUES FOR BIOMARK ER DISCOVERY BY SHOTGUN METHODS

G.Y. Monroy Núñez2, J.P. Reyes Grajeda1, K. Calderón-González1, J.L. Gallegos-

Pérez1.

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 formalin-fixed 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.


69

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.

1Posgrado 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 sexually-transmitted 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.


70

P08

PROTEMIC ANALYSIS OF ZEBRAFISH EMBRYOS EXPOSED TO T HE ENDOCRINE DISRUPTOR 17-αααα-ETHINYL-ESTRADIOL

Teresa Gutiérrez Espíndola1,2, Ernesto Maldonado Olvera2, Karla Grisel Calderón

González3, Nora Andrea Gutiérrez-Nájera3, José Luis Gallegos Pérez3

1CEACA,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.


71

P09

PROTEOMIC ANALYSIS OF EFFECT OF PROSTAGLANDINS ON M IDGUTS OF THE DENGUE VECTOR MOSQUITO Aedes aegypti

Borbolla-Vázquez J1., Cázares-Raga FE1., García-Gil de Muñoz FL2., Sánchez-Hernández H1., Cortés-Martínez L1., Rodríguez MH3, Hernández-Hernández FC1.

1Depto. de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, México, D.F. 2Universidad 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.


72

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.

aDepto. 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 database-predicted 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.


73

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

1Unidad de Proteómica, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, México

2Lab. 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 RP-HPLC, 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.

Acknowledge: This work was partially financed by grants from DGAPA IN-221508 to CVFB.


74

P12

PROTEOMIC ANALYSIS OF THE SKIN SECRETION OF THE MEX ICAN FROG LITHOBATES PUSTOLOSUS

Oscar Villa-Hernández1, Ruben Castro Franco2, Cesar V. F. Batista1

1Instituto 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.

Acknowledge: This work was partially financed by grants from DGAPA IN-221508 to CVFB.


75

P13

CARACTERIZACIÓN DE LA RESPUESTA INMUNE HUMANA CONTR A 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 SDS-PAGE 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


76

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.


77

P14

MASS FINGERPRINTING OF Centruroides limpidus tecomanus (BUTHIDAE SCORPION) VENOM AND BIOLOGICAL ACTIVITY OF VENOM FR ACTIONS

L. L.Valdez-Velazquez1, L. J. G. Zamora- Pizano1, F. I. Coronas2, F.Z. Zamudio2, C. Balderas2, L.D. Possani 2

1Facultad 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.


78

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.



79

P16

PROLINE SUBSTITUTION REDUCES THE HEMOLYTIC ACTIVITY OF A SHORT ANTIMICROBIAL PEPTIDE

Alexis Rodríguez1, Elizabeth Aguilar2, Elba Villegas2, Gerardo Corzo1

1Instituto de Biotecnología, Universidad Nacional Autónoma de México, 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 α-helical 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


80

P17

IDENTIFICATION AND ANALISIS OF THE DIHYDRODIOL DEHY DROGENASE (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).


81

P18

IDENTIFICATION OF DGKζζζζ-SNX27 INTERACTION: NEW COMPONENTS OF VESICULAR TRAFFICKING IN T LYMPHOCYTES.

Esther Rincón1, Teresa Santos-Mendoza1,2, Antonia Ávila-Flores1 and Isabel

Mérida1.

1Department of Immunology and Oncology, National Center of Biotechnology, Madrid, Spain. 2 National Institute of Respiratory Diseases, Mexico City, Mexico.

Email: [email protected]

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. Co-immunoprecipitation 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.


82

P19

PROTEIN EXPRESSION PROFILE IN PLASMA OF OBESE CHILD REN

Esparza-Ibarra Montserrat1, Flores-Altamirano Fátima1, Pérez-Luque Elva1, Garay-Sevilla Ma. Eugenia1, Macías-Cervantes Maciste H1, Encarnación Sergio2 y Pérez-Vázquez Victoriano1

1División 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.


83

P20

PROTEOMIC ANALYSIS OF MIXED BIOFILM Candida albicans -

Pseudomonas aeruginosa.

Trejo-Hernández A1., Andrade Domínguez A., Hernández Ortiz M., Encarnación Guevara S.M.

Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, 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.



84

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. 1Genomics 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.


85

P22

LYT1 OLIGOMERIZE UNDER ACIDIC CONDITIONS: IMPLICATIONS IN THE TRYPANOSOMA CRUZI INFECTION PROCESS

C. Lugo-Caballero1, G. Ballesteros-Rodea1, A. Rojo-Domínguez2, R. Manning-

Cela1. 1Molecular Biomedicine Department, CINVESTAV, Zacatenco, México.

2Departamento 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.


86

P23

RHIZOBIUM ETLI PHOSPHOPROTEOME DURING FREE LIFE AND SIMBIOSYS METABOLISM.

Magdalena Hernández-Ortiz, Angel Gabriel Martínez-Batallar, Sandra Contreras-Martínez, Agustín Reyes-Pérez, Yolanda Mora, and Sergio Encarnación.

Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México. Email: [email protected]

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 two-dimensional 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.

Part of this work was supported by CONACYT grant 60641 and DGAPA-PAPIIT grant IN222707


87

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.

1Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, 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 IN-222707 grant.


88

P25

ANALYSIS OF PROTEIN PROFILES OF ANTIBIOTIC-RESISTAN T Salmonella Typhimurium

Lugo-Melchor OY1, Barba de la Rosa AP2, Fagersquist CK3, Leon-Felix J1 and

Chaidez C1

1Centro de Investigación en Alimentación y Desarrollo, Culiacan, Sinaloa, México, 2Instituto 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 (LC-MS/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 antibiotic-resistance 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.


89

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, Gutiérrez-Nájera N.4 & Castañón-Arreola M.1

1Genomic 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.


90

P27

OPTIMIZATION OF A PROTEIN EXTRACTION METHOD SUITABL E FOR A FERMENTED MAIZE DOUGH PROTEOMIC STUDY

Cárdenas M. C.1, Delgado L1., Wacher C.2, Barkla B.3 and Rodríguez-Sanoja R.1

1Instituto 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.


91

P28

SHOTGUN PROTEOMICS AS A STRATEGY FOR THE CHARACTERI ZATION 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

aPlant 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 data-dependent 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 4-weeks 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.


92

P29

SEPARATION OF CALCIUM-DEPENDENT PROTEIN KINASES FRO M BEETROOT PLASMA MEMBRANES ACCORDING TO THEIR HYDROP ATHY,

AND THEIR IDENTIFICATION BY MASS SPECTROMETRY

M.B, Lino Alfaro1, A. Chagolla López2, F.C. Alcántar Aguirre1 L.E. González de la Vara1

1Departamento 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.


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P30

LOSS OF STARCH BIOSYNTHESIS ENZYMES DURING THEIR EX TRACTION 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 granule-associated proteins, even though the starch has not been gelatinized.


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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

1CINVESTAV-IPN, Irapuato, Guanajuato, México 2CORBANA, 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 well-known 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.


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P32

COMPARATIVE PROTEOMICS OF CHLOROPLAST BIOGENESIS AF FECTED Arabidopsis thaliana MUTANTS

A. Arturo Guevara-García1, Gabriel Martínez Batallar2, Maricela Ramos-Vega1,

Odette Avendaño Vázquez1, Carolina San Román1, Patricia León1 & Sergio Encarnación Guevara2

1Instituto de Biotecnología, Universidad Nacional Autónoma de México, 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 chloroplast-encoded 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.


96

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.

1Instituto 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


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PAPAYA FRUIT β-GALACTOSIDASE: A TWO-DIMENSIONAL GEL ELECTROPHORESIS STUDY

Lino-López Gisela Jareth1, Barba de la Rosa Ana Paulina2, Osuna-Castro Juan Alberto1

1Laboratorio 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 1-methylcyclopropene (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 two-dimensional PAGE (2-DE-PAGE), and its corresponding Western blot. Also the β-Gal 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.


98

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.

1Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima. Colima, México.

2Instituto 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.


99

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. Lanz-

Mendoza3, G. Hurtado-Sil3 & L.A. Bello-Pérez1

1Centro de Desarrollo de Productos Bióticos,Instituto Politécnico Nacional

2Instituto 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.


100

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, Barrera-

Saldaña, H.A. 1

1Departamento 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.


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P38

IMPLEMENTATION OF A MALDI-IMAGING PLATFORM IN RAT L IVER 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

1Instituto 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 MS-signals. 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-4-hydroxycinnamic 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.


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P39

IN DEPTH, COMPREHENSIVE MAPPING OF THE HUMAN SEMINA L PLASMA PROTEOME BY A NOVEL, ITERATIVE LC-MS/MS/DATA BASE

SEARCH WORKFLOW

Claire Dauly1, Antonie D. Rolland1, Martin Hornshaw2, Regis Lavigne3, and Charles Pineau2,3

1Thermo 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 LC-MS/MS and exclusion lists.

Methods : A list of peptides which were confidently identified from the first LC-MS 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).


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P40

INTACT PROTEIN SEQUENCING USING ETD AND PTR IN A DU AL-PRESSURE LINEAR ION TRAP

Zhiqi Hao1, Jae C. Shwartz1 and Andreas FR Huhmer1

1Thermo 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.


104

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.


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P41

QUANTITATIVE PROTEOMIC APPROACHES FOR THE DETERMINA TION 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

2Thermo 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.


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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, Talamás-

Rohana P.1

1Departmento 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.


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P43

ANALYSIS BY 2D-ELECTROPHORESIS AS A PREELIMINARY ST EP 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

1Centro de Investigación y de Estudios Avanzados, I.P.N., México D.F.

2Instituto 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 2D-gels.


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ANÁLISIS PROTEÓMICO COMPARATIVO DE TUMORES ONDONTOG ÉNICOS HUMANOS

García-Muñoz A.1, Cázares-Raga FE1., Hernández-Hernández FC1., Bologna-Molina R2., Rodriguez M.A1.

1Depto. de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, México, D.F. 2Facultad de Medicina, Universidad de Guadalajara, Guadalajara, Jalisco Hospital

Juárez de México. email: [email protected]

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.


109

P45

A PROTEOMIC ANALYSIS OF THE SECUNDARY EVENTS ASSOCI ATED WITH THE APPLICATION OF SCORPION TOXIN Cn2 ON EXCIT ABLE CELLS

Pedraza Escalona, MM1, Restano-Cassulini, R1., Batista, C.V.F1., Possani LD1.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, 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


110

ÍNDICE DE AUTORES

A

Acosta-Pérez M L38 57

Acuña-Askar K P37 100

Agis-Juárez R L08 19

Agama Acevedo E P30 93

Aguado-Santacruz A L16 28

Aguilar E P16 79

Aguilar-Vera A P24 87

Aillet F L13 25

Alcántar Aguirre FC P29 92

Alcaráz Flores S L08 19

Alvarez S L11 23

Alvarez-Sánchez ME P04, P07 66, 69

Amador-Gaytán E P13 75

Andrade Domínguez A P15, P20, P21 78,83,84

Angel-Ortiz C L33 52

Arroyo R L33, L42, P05, P07 52,61,67,69

Ascacio-Martínez JAI P37 100

Avendaño-Vázquez O P32 95

Ávila-Flores A P18 81

Avila-González L L33, L42 52,61

B

Baginsky S L35, P28 54,91

Balderas C P14 77

Ballesteros-Rodea G P22 85

Barba de la Rosa AP L35, L37, L41, P25, P28, 54,56,60,88,91

P33, P34, P35, P36 96,9798,99

Barkla BJ L15, P27 27,90

Barrera-Pacheco A P33 96

Barrera-Saldaña HA P37 100

Bárzana E P38 101

Batista CVF L03, L38, P11, P12, P45 14,57,73,74,109

Bautista-de Lucio VM L32 51

Bello Pérez LA P36 99

Benitez O L12 24

Bernhardt J L23 38

Bhoumik A L04 15


111

Bischof S L35, P28 54,91

Bologna-Molina R P44 108

Borbolla-Vázquez J P09 71

C

Calderón-González K L07, L31, P06, P08, P38,P42 18,50,68,70,101,106

Caprioli R L02, L05 13,16

Cárdenas MC P27 90

Cárdenas-Guerra RE L33 52

Castañón-Arreola M P07, P26 69,89

Castillo-Rodal AI P13 75

Castro Franco R P11, P12 73,74

Cázares-Raga FE L29, P09, P10,P44 48,71,72,108

Cázarez F P42 106

Chaerkady R L06 17

Chagolla López A L16 , L17, P29 28,30,92

Chaidez C P25 88

Chavez-Salinas S L08 19

Checa Rojas A L09 20

Chen H L06 17

Chen S L11 23

Cole RN L06 17

Contreras Martínez S L25, L34, L40, P23 42,53,59,86

Coronas FI P14 77

Cortéz-Martínez L L29, P09 48,71

Corzo G P16 79

Cruz-Colín JL L31 50

Cruz-Hernández A L36 55

Cuellar-Olalde R P35 98

D

Damían-Zamacona S L07 18

Damoc NE P41 105

Dauley C P39 102

De León-Rodríguez A L37 56

del Angel RM L08 19

Delgado L P27 90

Díaz-Tufinio C L31 50

E

Elizalde M L34 53


112

Easterling ML L20 34

Elortza PF L13 25

Encarnación Guevara S L09, L25, L30, L34, L40, P03, P15, 20,42,49,53,59,65,78

P19,P20, P21, P23, P24, P32,P46 82,83,84,86,87,95,110

England L13 25

Escobar-Tovar L P31 94

Esparza-Ibarra M P19 82

Espitia-Pinzón CI L26 44

F

Fagersquist CK P25 88

Ficarro SB L04 15

Flores-Altamirano F L30 ,P19 49,82

Flores-Pérez EC L30 49

Fonseca-Sánchez MA P04 66

G

Gallardo-Rincón D P43 107

Gallegos-Pérez JL L07, L31, P06, P08, P38, P42 18,50,68,70,101,106

Galván I P42 106

Garay-Sevilla ME P19 82

Garbis SD P41 105

García Gil de Muñoz FL P09, P17 71,80

García-Muñoz A P44 108

Garfias Y L32 51

Garibay-Cerdenares OL P43 107

Garibay-García JA P10 72

Gerrits B L35 54

Giannopolou EG P41 105

Gil C L41 60

Gimeno M P38 101

Goldsmith J L11 23

Gómez-Lim M P31 94

González de la Vara LE L17, P29 30,92

González -Zamorano M L26 44

Gucek M L06 17

Guerrero-Rangel A L16 28

Guevara-García AA P32 95

Gutierrez GJ L04 15

Gutiérrez-Espíndola T P08 70

Gutiérrez-González LH P01 63

Gutiérrez-Nájera N L31, P08, P26 50,70,89

Guzmán M P31 94


113

Guzmán Bautista ER P42 106

H

Hao Z P40 103

Hardesty W L05 16

Hernández M L09, L25, L34 20,42,53

Hernández G R L39 58

Hernández Orihuela L P11 73

Hernández Pando R P26 89

Hernández-Coronado M L15 27

Hernández-Estrada MG P17 80

Hernández-Hernández FC L29, P09, P10, P44 48,71,72,108

Hernández-Ortiz M P03, P20, P23, P24 65,83,86,87

Hernández-Ramírez VI P42 106

Herrera Y L25 42

Herrera-Carrillo Z P02 64

Herrera-Salgado Y L34, L40 53,59

Higareda V L12 24

Higareda Almaraz JC L09, P03 20,65

Hjerpe R L13 25

Hornshaw M P39, P41 102,105

Hoving S L23 38

Huerta-Ocampo JA L37, P33 56,96

Huhmer AFR P40 103

Hurtado-Sil G L38, P36 57,99

I, J, K

Izquierdo J L12 24

Javier Reyna R P42 106

Jianjung H L06 17

Jiménez Corona A L07 18

Juárez Garcia E P30 93

Kaspar S L01 12

Kim MS L06 17

L

Lang V L13 25

Langridge J P01 63

Lánz-Mendoza H L12, L38, P17,P36 24,57,80,99

Lavigne R P39 102

León P P32 95

Leon-Felix J P25 88


114

Lino-Alfaro MB L17, P29 30,92

Lino-López GJ P33, P34 96,97

Lloret-Sánchez L P13 75

López Luna A P38 101

López-Briones S L30 49

López-Camarillo C P04 66

López-Espinosa NL L32 51

López-Vidal Y L24, P13 41,75

Lopitz-Otsoa F L13 25

Lugo-Caballero C P22 85

Lugo-Melchor OY P25 88

Luna JP P04 66

Luviano-Bazán D L38 57

M

Macias-Cervantes MH P19 82

Maldonado Olvera E P08 70

Manning-Cela R P22 85

Mares-Álvarez D L30 49

Marjan G L06 17

Márquez-Ipiña AR P37 100

Martínez AG L34 53

Martínez-Barnetche J L38 57

Martínez-Batallar AG L25, P23, P24, P32 42,86,87,95

Martínez-Obregón F L40 59

Martínez-Salgado JL L41 60

Mas Oliva J L07 18

Matros A L01 12

McKenna T P01 63

Mendoza G L39 58

Mendoza Hernández G L25, L26, L32, L37, P04, P21, P26 42,44,51,56,66,84,89

Meneses Moreno N L25, P15, P21 42,78,84

Menezes EP P11 73

Mérida I P18 81

Min-Sik K L06 17

Mock HP L01,L18 12,31

Moehring T P41 105

Monroy Nuñez GY P06, P38 68,101

Mora Y L25, P23 42,86

Morales-Vásques F P43 107

Moreno A L07 18


115

O, P, Q

Ortega-López J L33 52

Ortiz-Plata A L29 48

Osorio C P43 107

Osuna-Castro JA P33, P34, P35 96,97,98

Pacheco Vargas G P30 93

Pandey A L06 17

Pando-Robles V L12 24

Pantoja O L15 27

Paredes-López O L36 55

Pascual N L04 15

Pedraza-Escalona MM P45 109

Pérez Carreón JI P38 101

Pérez-Luque E P19 82

Pérez-Vázquez V L30, P19, 49,82

Peters EC L04 15

Pimienta G L04, L06 15,17

Pineau C P39 102

Pitarch A L41 60

Possani LD P14,P45 77, 109

Prieto Conaway MC L19,L21 32,35

Quintanar Jurado V P38 101

Quintas Granados LI P07 69

R

Ramírez R P L39 58

Ramírez Torres AC L09 20

Ramírez-Emiliano J L30 49

Ramon-Gallegos E P02 64

Ramón-Luing LA L33, L42, P05 52,61,67

Ramos-Vega M P32 95

Regalado Santiago DT P38 101

Reiland S L35, P28 54,91

Rendón Gandarilla FJ L33, L42, P05 52,61,67

Resendis O L25 42

Restano-Cassulini R P45 109

Reyes del Valle J L08 19

Reyes-Grajeda JP L07, P06 18,68

Reyez-Perez A P23, P24 86,87

Reyna R P42 106

Rincón E P18 81

Robert C L06 17


116

Rodríguez A P16 79

Rodríguez MA P44 108

Rodríguez MS L13 25

Rodríguez MH P09, P10 71,72

Rodríguez Ambriz SL P36 99

Rodriguez Sanoja R P27 90

Rodríguez-Cuevas S P04 66

Rojo-Domínguez A P22 85

Rolland AD P39 102

Romero Martínez SA P15 78

Ronai Z L04 15

Rosas-Cárdenas FF L36 55

Roschitzki B L35, P28 54,91

Roumeliotis T P41 105

S

San Román C P32 95

Sánchez-Hernández H L29, P09 48,71

Sandoval J P31 94

Santos-Mendoza T P18 81

Saraswati S L06 17

Scigelova M P41 105

Seiffert U L01 12

Serrano-Pinto VV L38 57

Shwartz JC P40 103

Sierra-Martínez P P10 72

Silva-Sánchez C L14 26

Stegemann J L23 38

Strul JM L11 23

Sukumar S L06 17

T, U

Talamás-Rohana P P42,P43 106,107

Terán LM L22, P01 37,63

Torres-Ramos M L13 25

Trejo-Hernández A P15, P20 78,83

Utrilla Coello RG P36 99

V

Valdés-Rodríguez SE L16 28

Valdez-Velázquez LL P14 77

van Oostrum J L23 38


117

Vargas Lagunas MC L25, P24 42,87

Vargas-Romero F P26 89

Vázquez-Carrillo LI P07 69

Ventzki R L23 38

Vera-Estrella R L15 27

Villa-Hernández O P11, P12 73,74

Villanueva González P P38 101

Villegas E P16 79

Vissers H L27 45

W,X Y, Z

Wacher C P27 90

Watson JT L10, L28 22,46

Weier D L01 12

Weschke W L01 12

Witzel K L01 12

Xolalpa W L26 44

Xu S L06 17

Yocupicio Monroy M L08 19

Yien G 106 17

Zamora-Pizano LJG P14 77

Zamudio FZ P14 77

Zenteno E L32 51

Zhu M L11 23

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