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olfactory mucus give evidence of secreted
odorant-binding proteins
Loïc Briand, Benoît Sarazin, Celine Henry, Jean-Claude Huet, Corinne Eloit,
Didier Trotier, J Claude Pernollet
To cite this version:
32.12
A Unique Approach to Automated,
Parallel, High Throughput Protein
Expression and Purification
Linda E. Cammish
Director of Business Development, NextGen Sciences Ltd., Building 56, Alconbury North Airfield, Alconbury, Huntingdon, Cambs., PE28 4DA, United Kingdom
The role of proteomics research in disease identification and in the drug discovery process continues to rapidly expand. This is resulting in an ever-increasing need for new and improved tools to produce many hun-dreds of proteins, with relevant molecular diversity, for use in a whole range of different applications. These include high throughput screening, antibody production and characterisation, protein microarray applications and crystallography.
In this presentation a novel combination of tools, which include hard-ware, software and biochemistry, will be described that enable the whole process of protein expression and subsequent purification to be auto-mated. This system solution provides for the systematic, parallel produc-tion of many hundreds of purified proteins with minimum input and hands-on time. A range of unique expression vector systems will also be described that incorporate a series of fusion partners, enabling optimal protein expression of any protein from any organism, together with a choice of affinity tags for subsequent purification via native or denaturing purification protocols. The presentation will illustrate how it is possible to start with cDNA clones and fully automate the whole process of recom-binatorial cloning followed by expression and purification of hundreds of proteins in parallel for use in a whole range of different proteomics re-search applications. The platform technology incorporates intelligent sam-ple tracking from the starting cDNA clones and vector constructs right through to the final expressed and purified proteins.
33.1
Analysis of Posttranslational Modifications
of
␣-A-Crystallin During Aging of the Eye
Lens
Heike Schaefer1, Albert Sickmann1, Marion Herrmann2, Joachim Klose2, and Helmut E. Meyer1
1
Med. Proteom-Center, Ruhr-Universitaet Bochum, Germany; and
2Institut fuer Humangenetik, Charite, Humboldt Universitaet Berlin,
Germany
The eye lens allows to focus pictures on the retina. It consists of two types of cells, epithelial and fiber cells (1). The lens grows throughout life, maintaining transparency without significant turnover of its densely packed proteins (2). Mainly the crystallins are responsible for its transpar-ency. They comprise more than 90% of the lens proteins. During aging these proteins are posttranslationally modified, i.e., phosphorylated. We report the identification of different posttranslationally modified ␣-A-Crys-tallin after 2D-PAGE described by Klose et al. (3) of eye lenses (mus musculus) of different ages. The number of detected protein spots of ␣-A-Crystallin in the gel raised from 3 protein spots in the embryo state to 22 protein spots in the 2D-gel of the lens proteins analyzed from 100 week old mice. This let to the assumption that the number of modifications in the ␣-A-Crystallin accumulates with age. Using nano-LC-MS/MS ␣-A-Crys-tallin could be identified with a sequence coverage 50% in all protein spots. Furthermore, some posttranslational modifications, like phospho-rylation of specific serine residues could be detected. A set of these protein spots was also analyzed by MALDI-TOF-MS. With these additional measurements the data of the LC-MS/MS could be verified and comple-mented. This work demonstrates that LC-ESI-MS/MS and MALDI-TOF-MS are powerful complementary tools for the identification of post-translational modifications from single 2D-PAGE protein spots.
1. Colvis, C. M. et al. (2000) Electrophoresis 21, 2219 –2227. 2. Jaenicke, R. (1994) Naturwissenschaften 81, 423– 429. 3. Klose, J. et al. (1995) Electrophoresis 16, 1034 –1059.
33.2
Computer-assisted Mapping of Antigenic
and Functional Regions in Hepatitis C
Virus Proteins
Boris Sobolev and Ludmila Olenina
V.N. Orekhovich Institute of Biomedical Chemistry of Russian Academy of Medical Sciences, Pogodinskaya Str. 10, Moscow 119992, Russia
33.3
Protein Changes Induced by IL-1
 in
Islets of Langerhans In Vitro Followed In
Vivo During Development of Diabetes in
Diabetes Prone BB Rats
Thomas Sparre1, Ulla Bjerre Christensen1, Peter Mose Larsen2, Stephen Fey2, Carsten Gotfredsen3,
Peter Roepstorff4, Allan Ertman Karlsen1, Flemming Pociot1, and Jørn Nerup1
1
Steno Diabetes Center, DK-2820 Gentofte, Denmark;2
Center for Proteome Analysis, DK-5230 Odense, Denmark;3Novo Nordisk,
DK-2880 Bagsvaerd, Denmark; and4
South Danish University, DK-5230 Odense, Denmark
Interleukin-1 (IL-1 is involved in the destruction of insulin producing  cells in the islets of Langerhans and Type 1 Diabetes Mellitus (T1DM). By 2-dimensional gel electrophoresis (2-DGE) of IL-1 exposed BB-DP islets in vitro, 82 protein spots out of 1.815 changed expression. Syngeneically transplanted islets under the kidney capsule in pre-diabetic BB-DP rats facilitate studies of protein expression changes during spontaneous dia-betes development in vivo.
Aim: To track these 82 IL-1 induced protein changes in vitro in trans-planted islets in vivo.
Methods: 200 neonatal BB-DP islets were transplanted under the kid-ney capsule of pre-diabetic BB-DP rats and removed after 7, 12, 23, 37, 48, 174 days or at onset of diabetes and labelled with [35S]-Methionine for
2-DGE. IL-1 induced expression changes in vivo were identified and compared to control islets and identified by MALDI. Transplants were examined for IL-1 mRNA by in situ hybridisation.
Results: In situ hybridisation showed IL-1 mRNA expression from day 7 onwards. All 82 protein spots were found in the transplants. Twenty-one of the 82 spots were not changed in vivo. Twenty-tree to 45 proteins changed expression over time in transplants, fewest in rats escaping diabetes. From 37 spots, proteins involved in energy production, protein synthesis, chaperoning, protein folding, signal transduction, differentia-tion, apoptosis were identified by MALDI.
Conclusion: IL-1 is expressed in the in the transplants and many (40 –55%) of the protein changes induced by IL-1 in vitro also occur in vivo suggesting a pivotal role for IL-1 in the pathogenesis of T1DM in this model.
33.4
Exploring the Cardiovascular Pathology by
Functional Genomics Techniques
Pei-Wen Wang1and Tai-Long Pan2
1
Department of Biological Science, National Sun Yat-sen University; and2School of Traditional Chinese Medicine, Chang Gung
University
Coronary artery disease (CAD) is one of the most important health prob-lems in modern society with high mortality and morbidity. CAD is the third leading cause of death in Taiwan. The cellular and biochemical pathology associated with cardiac dysfunction has been extensively characterized in different animal models; however, the molecular mechanisms associated with hypoxia and ischemic/reperfusion injury are not well defined.
Traditional approaches for studying gene expression are limited to the analysis of a small number of genes under a defined physiological condi-tion. Most gene expression studies are directed at a specific cellular pathway or under stress response. The factors studied included apoptosis factors, transcription factors, heat shock proteins, and antioxidant en-zymes. In order to develop a rational therapeutic strategy for these dis-eases, it is imperative to clarify the network regulation of molecular events in acute coronary syndrome and chronic CAD. These factors may be uncovered by using novel techniques of functional genomics such as DNA microarrays and proteomics; these techniques hold great promise for the study of complex biological systems with applications in molecular medicine.
Protein expression profiling (proteomics) will allow us to obtain a protein expression profile of hypoxia, ischemia/reperfusion. The use of microar-rays will provide us with gene expression profiles. The application of technology to facilitate these analyses, including 2D gels with mass spec-trometry of complex protein samples and bioinformatics will be discussed. Understanding patterns of expressed genes is expected to improve our knowledge of highly complex networks that cross communicate in hitherto unknown ways both in healthy and disease states.
33.5
Exploring the Pathophysiology of Liver
Fibrosis by Proteomics
Tai-Long Pan
School of Traditional Chinese Medicine, Chang Gung University, Tao-You, Taiwan 333
Fibrosis is a dynamic pathological process of excess deposition of extra-cellular matrix components in the body. Liver fibrosis and cirrhosis are the end result of majority of chronic liver insults and represent a common and difficult clinical challenge of worldwide importance.
Chronic injuries leading to liver fibrosis occurs in response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drug use and abuse, metabolic disease due to iron or copper overload, autoimmune attack of hepatocytes or bile duct epithelium, and congenital abnormalities. Fibrosis forming mechanism is a multifunctional process that involved several cell types, soluble factors and results from a dysregulation of the homeostatic mechanisms that maintain the liver ecosystem.
Analysis of molecular mechanisms underlying chronic liver injury is essential for the development of effective therapies against liver fibrosis. To acquire a deeper knowledge on this subject we have initiated proteome analysis of genetic or virus-induced fibrosis patient’s serum and tissue. The combination of 2-D PAGE and MALDI-TOF mass spectrometry can resolve the complex proteome and characterize selected individual pro-teins with respect to identity, quantity, and post-translational modifica-tions in these fibrosis candidate proteins of patient.
33.6
Novel Affinity Ligands for Complexity
Reduction of Serum or Plasma Samples
Prior to High Resolution Proteomic
Analysis
Mark R. Kavonian1, Elena Chernokalskaya1,
Malcolm G. Pluskal2, Myra Robinson2, Sara Gutierrez1, and Aldo M. Pitt1
1
Millipore Corporation, Life Sciences Division; and
2Proteome Systems Incorporated
Proteomic analysis of complex samples, such as plasma or serum is frequently influenced by the presence of high abundance proteins such as albumin or immunoglobulin. These proteins can lead to loss of resolution in two-dimensional electrophoresis (2DE) and chromatographic separa-tions or excessive background noise in LC-MS/MS protein analysis meth-ods. As a result, sample complexity reduction to lower the level of these abundant proteins is rapidly becoming an essential first step of any high throughput analysis approach. Data will be presented to show applica-tions of affinity ligands for removal of abundant proteins from serum or plasma. The impact of this approach for removal of albumin and immu-noglobulin is assessed using an integrated Proteome analysis technology platform, employing 2DE analysis. Removal of these two major compo-nents was shown to considerably improve the detection of low abundance proteins in the resulting 2DE gels. The sample complexity reduction tech-nology described in this presentation has resulted in a kit employing small volume centrifugal devices. This kit format provides a convenient and efficient process for removal of abundant proteins from a complex sample, such as plasma or serum.
33.7
Proteins Involved in Recruitment of
Renin-expressing Cells During
Renovascular Hypertension
Identified by Proteomic Analysis
Florence Pinet1, Florence Poirier2, Naima Imam-Sghiouar2, Jean-Pierre LeCaer3, Michel Caron2,
Raymonde Joubert-Caron2, and Pierre Corvol4
1
INSERM U508, Lille, France;2
Laboratory of Proteins and
Proteomics, Bobigny, France;3ESPCI, Paris, France; and4INSERM
U36, Paris, France
33.8
An Orthogonal High Resolution
Three-Dimension Separation System
Coupled with DIGE for Quantitative
Analysis of Serum Proteins
Hong Wang, Rong Zhao, Robert Hinderer, Dave Misek, and Sam Hanash
The University of Michigan, Medical Center, MSRB 1, Ann Arbor, Michigan 48109
Currently the standard approach for global proteome analysis consists of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with biological mass spectrometry (Bio-MS). While the 2D-gel approach has several advantages, the limited dynamic range and ability to resolve small and large proteins represent important drawbacks. The problem of dynamic range is particularly accentuated for biological fluids such as serum in which proteins vary in abundance over a 1,000 to 1,000,000 fold range with most potential biomarkers occurring in the low abundance range. We have developed an orthogonal high resolution three-dimen-sional (3-D) protein separation system to probe the serum proteome. This system separates protein based on charge, hydrophobicity and molecular mass. Serum samples are labeled with dyes prior to mixing and separation using DIGE reagent. The coupling of this approach with Bio-MS, such as MALDI-TOF MS and ESI Q-TOF MS/MS has led to the identification and quantitative analysis of a large number of low abundance proteins previ-ously not reported to occur in serum including Wiskott-Aldrich syndrome protein family member 1 (WASF1) which is involved in the transmission of signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton; a protein previously identified as cytosolic ovarian carci-noma antigen, and glial fibrillary acidic protein (GFAP) which has been considered as a cell-specific marker which distinguishes astrocytes from other glial cells during the development of the central nervous system. The 3-D system provides a novel approach for the comprehensive profiling, quantitative analysis and databasing of proteins in biological fluids.
33.9
Two-dimensional Mapping of Cellular
Prion Protein from Cerebrospinal Fluid
and Central Nervous System:
Immunoblotting and Purification Via
Dedicated Antibodies
Natascia Campostrini1, Annalisa Castagna1,
Alessia Farinazzo2, Gianluigi Zanusso2, Salvatore Monaco2, and Pier Giorgio Righetti1
1
Department of Agricultural and Industrial Biotechnologies, University of Verona, Verona, 37134, Italy; and2Department of
Neurological and Visual Sciences, University of Verona, Verona, 37134, Italy
The cellular prion protein (PrPC) is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein abundant in neurons. PrPC represents the sub-strate for the generation of a conformational pathogenic isoform (PrPSc) in human and animal prion diseases. By applying novel solubilization cock-tails, we analyzed normal human brain and cerebrospinal fluid (CSF) PrPC by 2-D mapping and immunoblots, using specific antibodies. Here we show that PrPC, from brain and CSF, is composed of several charge isomers of differently glycosylated isoforms of the full-length PrPC and some N-terminally truncated fragments. We also succeeded in capturing PrPC from brain homogenates via specific antibodies grafted to silica surfaces and are currently analysing by mass spectrometry the various isoforms eluted from 2-D maps.
33.10
Isoenzyme Forms and Catalytic Activity of
Brain Na
ⴙ-K
ⴙ-ATPase During Ageing
Roberto Federico Villa and Antonella Gorini
Department of Physiol. Pharmacol. Sciences, University of Pavia, Piazza Botta, 11, 27100 Pavia, Italy
The catalytic properties of ATP-ases related to ions homeostasis were evaluated in different types of somatic (SM) and synaptic plasma mem-branes (SPM) from cerebral cortex of rats aged 5, 10, 22 months: Na⫹, K⫹-ATP-ase, Ca2⫹, Mg2⫹-ATP-ase, Mg2⫹-ATP-ase are involved in regu-lation of pre-synaptic nerve ending homeostasis and post-synaptic activities.
33.11
Characterization of the N-linked
Oligosaccharides of an Immunoglobulin
M 12A1 Protective Against Cryptococcus
neoformans
Ruth Angeletti, Fang Wang, Antonio Nakouzi, and Arturo Casadevall
Albert Einstein College of Medicine, Bronx, New York 10461
Immunoglobulin M (IgM) is an especially important product of the immune system and plays a critical role in early protection against bacterial infec-tions. Cryptococcus neoformans, is remarkable among the medically im-portant fungi because it has a large polysaccharide capsule, composed primarily of glucuronoxylomannan. Certain IgMs that bind glucuronoxylo-mannan are protective against C. neoformans. IgM glycosylation has been implicated in important biological functions including classical pathway complement activation and avidity. In this investigation, the glycosylation pattern of the mAb IgM 12A1 was analyzed by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. Five of the six potential N-glycosylation sites were utilized. Striking heter-ogeneity was observed, with more than 47 glycoforms identified. This underscores the challenges that will be encountered in proteomics studies of glycosylated proteins. Studies of the relationship of glycosylation pat-terns to biological function of IgMs are being continued.
33.12
Identification of Proteins Expressed in
Human Plasma Exposed with Benzene by
2D Gel Electrophoresis
Won-A Joo, Won-kyu Son, Kyungsuk Choi, Hyun-Ju Lee, Do-Youn Lee, and Chan-Wha Kim
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
Benzene is one of the solvents used most widely in chemical agents, paint and dye industries. Exposure to benzene is associated with hematotoxicity including aplastic anemia, acute myelogenous leukemia and lymphoma. The benzene biomonitoring method based on single marker such as urine phenol, S-phenylmercapturic acid, trans-trans muconic acid has been noted to be unreliable for total benzene expose test. Therefore, to search a new biomarker for benzene exposure, we investigated proteins which significantly expressed in plasma of benzene exposure group (n⫽ 50) using two-dimensional gel electrophoresis (2-DE). Protein expression pro-files in benzene exposure group was similar to that in normal group, however several spots showed higher expression in all benzene exposure group than in normal group (p⬍ 0.05).
The specific proteins for benzene exposure were identified by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) and con-firmed by western blot. T cell receptor chain, FK506-binding protein (FKBP51), and TRF2-interacting telomeric RAP1 protein were expressed in only benzene exposure group. In addition, interleukin-4 receptor␣ chain and angio-associated migratory cell protein were found to be uniquely overexpressed in plasma of benzene exposure group. The proteomic profiles of benzene exposure group vs. normal group may generate more directly important markers for early detection and/or therapeutic targets unique to the benzene expose. Furthermore, these results will improve our understanding for the risk estimation and of the biochemical mechanisms of benzene expose.
33.13
Metabolic Syndrome and Monocyte
Activity
Peteris Tretjakovs, Uldis Kalnins, Inese Dabina,
Andrejs Erglis, Inese Dinne, Antra Jurka, and Valdis Pirags
P. Stradins Clinical University Hospital, LV 1002 Riga, Latvia
Aim: in order to examine the hypothesis that thymidine phosphorylase, an angiogenic factor is expressed in atherosclerosis, we examined the effect of metabolic syndrome on the viability of peripheral blood monocytes in diabetics with coronary artery disease (CAD). Type 2 diabetic (NIDDM) patients with CAD were divided in two groups: with (A) or without (B) metabolic syndrome. 20 healthy controls were examined as controls (C). The groups were matched for age and sex. In A group, the patients were with good glycemic control without diabetic complications, adiposity and dyslipidemia. In B group, the patients were with insulin resistance, adi-posity, bad glycemic control, microalbuminuria and dyslipidemia. Mono-cyte viability was assessed by IL-2-mediated 1Ci [3H]-thymidine
incor-poration into the monocytes. Radioactivity incorporated in DNA was solubilized by 0.2M NaOH and determined by a liquid scintillation counter. Results: [3
H]-thymidine uptake was distinctively higher in NIDDM patients with coronary artery disease with metabolic syndrome compared to the controls (B vs C, p⬍ 0.001). In patients without metabolic syndrome, the difference in [3H]-thymidine incorporation was insignificant (p⬎ 0.05) in
33.14
Serum and Urine Proteomics: Protein
Profiling and Identification of Ovarian
Cancer Biomarkers Using Mass
Spectrometry and Liquid Chromatography
Bin Ye1, Samuel Mok1, Steven Skates1, Steven Gygi2, Lanfei Fu1, Ross Berkowitz1, and Daniel Cramer1
1
Brigham and Women’s Hospital, Harvard Medical School; and
2Cell Biology Department, Harvard Medical School
The objective of this study is to identify promising ovarian cancer protein/ polypeptide biomarkers from human serum and urine specimens. The combined technologies of Surface Enhanced Laser Desorption (SELDI)-Mass spectrometry, Liquid Chromatography (LC) and Ion Trap Tandem Mass Spectrometry (MS/MS) have been applied in this study. Total of serum (203) and urine (103) subjects were collected from normal health women and ovarian cancer patients. In the serum proteomic studies, five protein peaks (m/z: 5400, 8929, 11723, 12410, 14141) distinguished the cancer patients from the normal controls in the IMAC chip profiles. In the urine proteomic studies, one protein peak (m/z:17407) of WCX profile appeared with a higher intensity in cancer patients. Two potential interest proteins in serum (m/z: 5400, 11723) and one in the urine (m/z: 17407) have been further purified with liquid chromatography. The purified proteins have been first verified by the SELDI-MS to confirm the mo-lecular weight or m/z. The proteins then were isolated from SDS-Gel and the tryptic peptides and amino acid sequence were determined with tandem mass spectrometry (MS/MS). The protein identities of 5400, 11723, 17407 correspond to calmodulin-like skin protein (CLSP), Hap-toglobin␣ chain (Hp-␣), and eosinophil-derived neurotoxin (EDN), re-spectively. Specific polyclonal antibodies against Hp-␣ and EDN were applied in western blots and ELISA to confirm their potential as ovarian cancer biomarkers in serum and urine. We conclude that proteomic studies of human serum and urine with mass spectrometry are powerful approaches for preliminary screening and identification of potential cancer biomarkers.
33.15
Proteomic Analysis of a Mammalian
Model of Parkinson’s Disease Using
2D-DIGE-MS
Judith Pickering1*, Varsha Godbole1, Sue Fowler1, Steve Lewis1, Karl Sko¨ld2, Marcus Svensson2, and Per Andren2
1
Amersham Biosciences UK Limited, Amersham, Buckinghamshire, HP7 9LL, United Kingdom; and2Uppsala University, Uppsala,
Sweden
The identification of protein abundance differences as a consequence of disease progression is critical to understanding the mechanics of disease. The integration of two-dimensional gel electrophoresis (2-DE) with mass spectrometry, remains the benchmark technology for complex protein separation, identification and characterisation.
This study has utilised the integrated Ettan™ (Amersham Biosciences) Proteomics Platform to investigate protein abundance differences in a mammalian model of Parkinson’s disease. The platform comprises the Ettan DIGE labelling technology for protein difference analysis, the Ettan Spot Handling Workstation for the automated manipulation of proteins for spot picking, digestion and spotting, and the Ettan MALDI-ToF Pro Mass Spectrometer for protein identification.
Parkinson’s disease (PD) is a neurodegenerative disorder characterised by tremor, rigidity and poor balance. Treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is one of several models used to mimic PD in humans. Administration of MPTP leads to the production of 1-methyl-4-phenyl-2,3-dihydropyridinium (MPP(⫹)). MPP(⫹) is taken up by dopamin-ergic neurons, causing mitochondrial dysfunction and cell death.
This study used 2D-DIGE-MS to investigate the changes in protein abundance within the striatum at different time-points following adminis-tration of a single dose of MPTP.
* Tel: ⫹44 1494 543414; Fax: ⫹44 1494 543278; Email: Judith. Pickering@uk.amershambiosciences.com
33.16
Comparative Proteomic Exploration of a
Human Mitochondrial tRNA Disorder
P. Tryoen-Toth1, S. Richert2, B. Sohm1, A. Van Dorsselaer2, E. Leize2, and C. Florentz1
1
UPR 9002, Institut de Biologie Mole´culaire et Cellulaire du CNRS, 67084 Strasbourg Cedex, France; and2Laboratoire de
Spectrome´trie de Masse Bio-Organique, Ecole de Chimie des Polyme`res, 67087 Strasbourg Cedex 2, France
MERRF sydrome (Myoclonic Epilepsy with Ragged Red Fibers) is a neu-romuscular disorder correlated to a single point mutation in a mitochon-drial gene coding for a transfer RNA, tRNALys
33.17
Proteomic and Glycomic Analyses of
Mouse Uterine Luminal Fluid
Chu-Wei Kuo1, He-Hsuan Hsiao2, Wei-Tzer Shyu2, Chin-Mei Chen1, Ying-Chu Lee1, Sin-Tak Chu1, and Kay-Hooi Khoo1,2
1
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; and2Core facilities for Proteomic Research, Academia Sinica,
Taipei, Taiwan
The accumulation of uterine luminal fluid (ULF) in the progestrus phase of rodent reproductive cycle is a well known biological phenomenon which can be simulated experimentally by estrogen stimulation. Only a limited number of proteins in the mouse ULF have previously been identified and investigated at the structure and function level. We sought here to derive a proteomic map of mouse ULF, its quantitative variation over different doses of estrogen stimulation, the post-translational modifications con-tained within, their interactions and functional implications in reproductive biology especially in mediating the maturation of sperm in uterus. Direct nanoLC-MS/MS approach on the total tryptic peptides derived from ULF has led to identification of numerous proteins not previously known to be present in ULF. Notably, a number of degradative enzymes are detected together with several involved in innate immunity. Parallel 2D gel/MS approaches revealed that most of the proteins present themselves as isoforms or glycoforms. Fucose-specific lectin blots confirmed that a majority of the proteins detected on gels are fucosylated glycoproteins. In addition to a previously characterized 24p3 known to modulate sperm activity, another major component, lactoferrin, was found to be likewise highly fucosylated. Mass spectrometry analyses led to identification of the glycosylation sites while further glycomic analyses on the released glycans from either individual proteins or the whole ULF proteome demonstrated a high abundance of complex type structures with sialylated antennae and/or Lewis epitopes. Co-purification by affinity binding indicated a potential interaction between these two proteins which share similar glyco-epitopes implicated in sperm motility.
33.18
Comparative Proteomic Investigations of
the Human Olfactory Mucus Give
Evidence of Secreted Odorant-binding
Proteins
Loı¨c Briand1, Benoıˆt Sarazin2, Ce´line Henry1, Jean-Claude Huet1, Corinne Eloit3, Didier Trotier4, and Jean-Claude Pernollet1
1
INRA F-78352 Jouy-en-Josas, France;2
Proteomic Solutions F-27950, St. Marcel, France;3Hoˆpital Lariboisie`re, F-75010 Paris,
France; and4
EPHE-CNRS, F-91305 Massy, France
33.19
Altered Protein Profiles of Postsynaptic
Density (PSD) by Ischemic Insults on Rat
Mi Hyun Kim1, Dong Hoon Kang1, Tae Hyuk Kang1, Eun Seok Seo1, Nak-Won Sohn1, Myungkoo Suh2, and Chulhun Kang1
1
Department of Neuroscience, Graduate School of East-West Medical Science, Kyung Hee University, Yongin-City, Kyungki-Do 449-701, Korea; and2
Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Korea
The postsynaptic density (PSD) is a specialized submembranous structure where neurotransmitter receptors are linked to cytoskeleton and signaling molecules. Morphological change in post-synaptic area is induced by cerebral ischemia. Therefore, characterization of the morphological change induced by ischemic insults in molecular levels should be essential for neuropathology. We isolated PSDs from ischemic brains of Sprague-Dawley rats where ischemic insults were induced by focal transient and permanent middle cerebral artery occlusion and constructed protein pro-files based on 2D electrophoresis. Over 300 protein spots were observed using G250 CBB-based staining. Interestingly, the global expression pat-terns are very similar each other regardless ischemic insults implying that the morphological changes of post-synaptic region associated with cer-ebral ischemia may not involve new protein members in large scale. Tubulin, actin, HSC 70, PKC, MAPKK and etc. were identified on gel. The expression levels of about 13 protein spots were altered by ischemic insults, however, these are very similar for both ischemic models with few exception.
This study was supported by 01-PJ9-PG1-01CO03-0003 and BK 21 project, Korea
33.20
Proteomics Study on the Function of
Simvastatin to the Artery of
Atherosclerotic Rabbit
Yan-ling Yu1,2, Peng-yuan Yang2, Hui-zhi Fan1,
Zhenyu Huang1, Yao-cheng Rui2, and Peng-yuan Yang1
1
Department of Chemistry, Fudan University, Shanghai 200433, China; and2Pharmaceutical College, Secondary Military Medical
University, Shanghai 200433, China
Aim: Proteomics study was applied to the evaluation of inhibitory effect of Simvastatin via investigating the overall expression level of proteins in the artery of the atherosclerotic rabbit.
Method: Experimental model was established by feeding the rabbits with a high fat diet (cholesterol 0.5g/kg/d, lard 0.5ml/kg/d) for 4 weeks; and then feeding them with Simvastatin (5mg/kg) for 8 weeks. The proteins were extracted from lytic cell of myocardial tissue. The overall protein levels were measured using two-dimensional gel electrophoresis and the resulted proteomic gel-spectra were analyzed by a PDquest data processing.
Result: Six runs showed a good reproducibility for two-dimensional gel electrophoresis. More than 2000 protein spots were detected. 29 protein spots showed significant quantitative changes in comparison of the nor-mal with the diseased rabbits, furthermore, the intensities of only a few spots were back-regulated after the diseased rabbit treated with Simvas-tatin, though an obvious decay of symptom of fatty liver was observed.
Conclusion: Simvastatin facilitates the metabolism of fat in the blood, but the lesion of artery internal wall of atherosclerosis cannot be restored.
33.21
Protein Changes in Thyroid Diseases
Jisnuson Svasti1,2, Chantragan Srisomsap1, Pantipa Subhasitanont1, and Phaibul Punyarit3
1
Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand;2Department of Biochemistry, Faculty of
Science, Mahidol University, Bangkok 10400, Thailand; and
3Department of Pathology, Pramongkutklao College of Medicine,
Bangkok 10400, Thailand
Thyroid nodules are common, but only a small number are eventually diagnosed as cancers. The most accurate and cost-effective method for the presurgical management of thyroid nodules is fine-needle aspiration biopsy (FNAB) but differential diagnosis of multinodular goiter, follicular adenoma and follicular carcinoma is sometimes difficult. We have studied the changes in proteomic patterns in different thyroid diseases, including normal thyroid (N), multinodular goiter (G), diffuse hyperplasia (Hy), follic-ular adenoma (FA), follicfollic-ular carcinoma (FC) and papillary carcinoma (PC). Four spots of cathepsin B were found with different expression in different thyroid diseases. Two of these cathepsin B spots, CB2 and CB3 are strongly up-regulated in neoplastic diseases (FA, FC and PC), compared to non-neoplastic diseases (G, Hy). The expression of galectin-3 was also studied in thyroid diseases by SDS-PAGE immunoblotting. Immunoreac-tivity was strong in PC but weak in N, Hy and almost absent in FA, FC and G. Only PC showed 2–3 bands of galectin-3. Two dimensional immunoblot analysis of galectin-3 in PC showed at least 3 spots with MW 34/pI 7.1, 32/7.5 and 30/7.5. Both cathepsin B and galectin-3 are useful for diagno-sis of multinodular goiter, follicular adenoma and follicular carcinoma.
Supported by the Chulabhorn Research Institute.
33.22
Identification of Cardiac Protein
Expression Abnormalities in Diabetes
Mellitus by LC-MS/MS
Lisa Stuart1,2, Allen Clermont2, Xiaohong Chen2, and Edward Feener2
1
Boston University, Boston, Massachusetts; and2
Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts
33.23
Tools for High-throughput Proteomics—
Expression Profiling for Diabetes
Research
Marcus Macht1, So¨ren Deininger1, Stefan Lehr2, Petra Palloch1, Stefan Mu¨ller3, Michaela Brodacki1, Armin Herkner2, Jo¨rg Kotzka2, and Dirk Mu¨ller-Wieland2
1
Bruker Saxonia Analytik GmbH, 04318 Leipzig, Germany;2
German Diabetes Center, 40225 Du¨sseldorf, Germany; and3University of
Cologne, 50931 Cologne, Germany
Diabetes mellitus type II and obesity are among the world’s most common diseases. The pathogenesis of both disorders is related to decreased insulin action and increased lipid accumulation in skeletal muscle, pan-creatic-cell, fat and liver. To investigate the impact of potential drug targets and transcription factors (e.g., SREBPs and PPARs) controlling cellular lipid metabolism as well as insulin sensitivity on gene expression and protein profiles, we are using the human liver cell line HepG2. For expression profiling, standardized human HepG2 cell lines have been established and analyzed by 2-DE using 24 cm ultra-zoom gels. The spots have been detected automatically using dedicated software and subse-quently picked by a robot. More than 1000 spots have been picked from each gel. The picked spots have been digested in an automated way in batches of 384 samples and a fraction of each sample was transferred onto MALDI-target plates. Each sample was analyzed by automatic ac-quisition of peptide mass finger print spectra as well as MALDI-TOF/TOF-spectra. The whole process flow was controlled by transponder chips to avoid mixing up of gels, sample plates or MALDI targets during the process. Spots which could not be identified by MALDI-MS alone were additionally analyzed by nanoLC-MS/MS. All data were transferred and stored in our data warehousing software ProteinScape for further analysis. This software allows for automated protein identification using different database search engines, detailed data analysis using sophisticated re-trieval functions, statistical data evaluation with third party software as well as further data retrieval in the WWW.
33.24
PARIS: An Proteomic Analysis and
Resource Indexation System
Juhui Wang1, Christophe Caron2, Alain Trubuil1, Michel-Yves Mistou3, and Christophe Gitton3
1
INRA, Biome´trie;2
INRA, Mission Informatique; and3
INRA, UBSP
Two dimensional polyacrylamide gel electrophoresis is currently the most popular method for analyzing proteins of living organisms. It allows ex-pression, characterization and separation of proteins on a gel substrate, according to their polarity and molecular weight. One of the challenges in this technology is to manage the massive amount of information. There are three major issues involved. The first is to keep track of the experiment information, the second is to process images to obtain the quantified protein expression value, and the third is to mine the information from the protein expression data. Although progress takes place every day on each issue, interconnecting the issues and exploiting disparate information and data emanating from different experiments or experimenters remains a hardly exploited topic. However, cross multi-experiment and multi-exper-imenter data validation might potentially yield tremendous benefits and assist us in forming more effective assessments about protein discovery and expression characterization. The goal of this paper is to present a general purpose proteomic analysis and resources indexation system called PARIS which makes the Internet an ideal tool for 2D gel data integration and sharing, quality improvement of protein expression profil-ing, and cross validation of disparate proteomic data.
Centered at a PostGreSQL database, PARIS integrates the above-mentioned three issues in a processing unit. It manages the information about experiments, gel images, protein expression and genomic resource and allows the user to search and analyze a large gel collection via Internet. It also supports visual verification of the analysis results and provides tools for advanced concept manipulation like virtual gel synthe-sis, protein expression prediction, implicit spots matching deduction, and cross multi-experiment, multi-experimenter data validation
33.25
The Analysis of Plasma Proteomics in
Trauma Mouse
Jianning Yin1, Yingxin Xu2, Xueying Gu1, Jingqiang Wang1, Rong Wang1, and Siqi Liu1
1
Division of Proteomics, Beijing Genetics & Bioinformatics Institute, Chinese Academy of Sciences, Beijing, China, 101300; and
2
Department of General Surgery, Beijing 301 General Hospital, Beijing, China
33.26
Spinal Cord Injury-associated Proteins in
Adult Rats Separated by Two-dimensional
Gel Electrophoresis and Identified by
Mass Spectrometry and Immunoblotting
Qinxue Ding1,2,3, Lin Xiao1, Yaojun Guo2, Fanwen Kong1, Shaoxiang Xiong3, Suqian Jing1, and Shaojun Liu1
1
Institute of Basic Medical Sciences, Beijing, 100850, People’s Republic of China;2Institute of Biophysics, Beijing 100101, People’s
Republic of China; and3
Institute of Chemistry, Beijing 100080, People’s Republic of China
Technological advances lead to more and better tools to address the inherent complexity of the adult mammalian central nervous system. In this paper, we successfully separate and identify a couple of differentially displayed proteins in the injured spinal cord of adult rats. Proteins in normal and injured spinal cords (complete transection at 8 –9 level of thoracic vertebrae for 5 days) were sequentially extracted with Tris buffer and enhanced solution containing 5 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 2% N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB3–10). Protein mixtures were separated by the two-dimensional gel electrophore-sis and visualized by Coomassie Brilliant Blue. Differentially displayed spots were identified by peptide mass fingerprinting and confirmed by immunobloting. Those unidentified by peptide mass fingerprinting were transferred for sequence analysis by tandem mass spectrometry. As a result, 20 up-regulated and 4 down-regulated proteins were screened out. Of those over-expression proteins, HSP27, GRP78, ERp60 and ERp29 are stress-induced; ubiquitin carboxyl-terminal hydrolase, proteasome, fatty acid binding protein and apolipoprotein are associated with nerval me-tabolization; connective tissue growth factor and follistatin-related protein are involved in wound healing and scar formation; Dynein, dynactin, an-nexin III, vimentin and GFAP are cell-growth and regeneration related. Roles of other up-regulated proteins in spinal cord injury, such as olfactory cyclic nucleotide channel and GABAA theta, cannot yet be assigned. Furthermore, one novel protein containing a peptide sequenced as VWCP-EGYCHEHK was found up-regulating in the injured spinal cord. Explora-tions of its whole length of cDNA and possible neurobiological roles are just under the way.
33.27
Proteome Study of the Protective Effect
Against Liver Ischemia/Reperfusion Injury
by Lidocaine Injection into
Hepatoduodenal Ligament in Rats
Mingyi Chen1, Chonghui Li1, Qinxue Ding2, Shaojun Liu2, and Zhiqiang Huang1
1
The Chinese PLA general hospital, Beijing 100853 , People’s Republic of China; and2Institute of Basic Medical Sciences,
Beijing 100850, People’s Republic of China
Objective: Ischemia/reperfusion (I/R) injury is still a problem in hepatobili-ary surgery. We have previously shown the data that 0.33% lidocaine injection into hepatoduodenal ligament prior to hepatic I/R could mimic hepatic ischemic preconditioning and exert some protective effects. This study aims to investigate related proteins to this novel approach.
Methods: Extracted proteins of liver tissues from the rats subjected to 40-min hepatic I/R injury with or without pretreatment of lidocaine were separated by two-dimensional polyacrylamide gel electrophoresis. Their gel maps were compared, those differentially expressed proteins were analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry.
Results: Ten proteins altered their expressions upon the lidocaine pre-treatment, of which seven proteins were up-expressed, including BID, FL-ATPase and glutathione synthetase; Three proteins were down ex-pressed, such as palmitoyltransferase, methylcrotonoyl-coenzyme A and carboxylase.
33.28
Peptidomics-based Discovery of Novel
Neuropeptides: Proteomics and
Peptidomics of a Parkinson’s
Disease Model
Per Andren1,2, Marcus Svensson3, Karl Skold3,
Judith Pickering4, Sven-Erik Skold2, and Per Svenningsson5
1
Uppsala University, SE-75124 Uppsala, Sweden;2
Amersham Biosciences, SE-75184 Uppsala, Sweden;3Uppsala University,
SE-75124 Uppsala, Sweden;4
Amersham Biosciences, Amersham, Buckinghamshire, HP7 9LL, United Kingdom; and5Karolinska
Institute, SE-17177 Stockholm, Sweden
Modern proteomic methodologies have significantly improved the possi-bilities of large-scale identification of proteins. However, these methodol-ogies are limited by their inability to reliably detect peptides. Here we describe an approach that has enabled us to detect more than 800 endogenously processed peptides in hypothalamic extracts in a single analysis. Automatic tandem mass spectrometry and amino acid sequence determination of 10% of these peptides showed that they consist of both novel and previously described neuropeptides processed from precursors such as proopiomelanocortin (POMC), cocaine and amphetamine regu-lated transcript, secretogranin I, proenkephalin A, neurosecretory protein VGF and stathmin. In addition, it was possible to identify several novel post-translational modifications of the neuropeptides. The combination of a simple peptide purification procedure, capillary nanoscale liquid chro-matography automatic switching tandem mass spectrometry and virtual image maps of endogenous neuropeptides is a powerful peptidomic ap-proach to investigate a large number of novel and known peptides and their post-translational modifications in complex tissues such as the brain. Further, utilizing the peptidomic approach as well as two-dimensional fluorescence difference gel electrophoresis (Ettan DIGE, Amersham Bio-sciences) we examined the change in protein and peptide expression in a time-course study of a mammalian model (mouse MPTP model) of Par-kinson’s disease. Changes in protein expression as low as 10% difference could be identified. Eleven proteins displayed a significant change after 1-day post MPTP treatment, 8 proteins after 8 days, and 15 proteins after 22 days post MPTP treatment in the striatum, respectively.
33.29
Global Proteomics for Human Blood
Serum I: Multidimensional Separation of
Peptides Coupled with Mass
Spectrometry
Joshua Adkins1, Susan Varnum1, Kenneth Auberry1, Ronald Moore1, Nicolas Angell2, David Wunschel1, Richard Smith1, David Springer1, and Joel Pounds1
1
PNNL, Richland, Washington 99352; and2
Human Genome Science, Rockville, Maryland
33.30
Alterations to the Myocardial Protein
Profile as a Consequence of
Ischemia/Reperfusion Injury
Melanie Y. White1,2, Stuart J. Cordwell2,
Hugh C. K. McCarron1, George Craft2, Adrian S. Tchen3, Brett D. Hambly3, and Richmond W. Jeremy1
1
Departments of Medicine, University of Sydney, NSW, Australia;
2Australian Proteome Analysis Facility, Macquarie University, NSW,
Australia; and3
Pathology, University of Sydney, NSW, Australia
The precise mechanisms that result in myocardium being reversibly dam-aged as a consequence of brief ischemia followed by reperfusion remain unresolved. Previous studies have suggested that the generation of oxy-gen free radicals (OFR) during reperfusion may be one possible mecha-nism. The relatively short time course for the onset of injury suggests that modifications to key proteins lead to the clinical manifestation. We have utilised proteomics to investigate stunning and furthermore we show that alterations to multiple proteins may be reversed by scavenging OFR. Left ventricular (LV) samples were taken from rabbit hearts after either 75 min control perfusion or 15 min low flow (1 ml/min) ischemia followed by 60 min reperfusion (15I/60R). A final group underwent 15I/60R, with OFR scavenger, N-(2-mercaptopropionyl) glycine (MPG) added (n⫽ 6/group). Isovolumetric LV pressure was measured throughout. Protein profiles were generated by two-dimensional electrophoresis (2-DE) and mass spectrometry to identify differentially abundant proteins. Rate pressure product at the end of the protocol was impaired in 15I/60R compared to control, consistent with stunning. Comparative analysis revealed 37 dif-ferences (⬎2-fold difference in expression) which can be categorised into 5 functional groups. These groups include sarcomeric proteins (e.g., MLC-2 fragmentation), cytoskeletal proteins (e.g.,␣-actinin 2) and ion transporting proteins (e.g., Na/K-ATPase). The addition of MPG was able to reverse these changes. Fragmentation of un-phosphorylated MLC-2 (15kDa) was shown to occur resulting in the removal of 15 residues from the N-terminus, immediately prior to the Ser16
phosphorylation site. The molecular mechanism of stunning is most likely a multifactorial process, involving damage to and/or alteration of several key protein systems. This study shows that scavenging of OFR results in the amelioration of some of these modifications.
33.31
Significant Coverage of the Normal
Human Serum Proteome by Gel-Less
LC-ESI-ION TRAP
John Marshall, Chris Smith, Weimin Zhu, Leslie Ingratta, Lisa Barker, Deborah Pinchev, Rulin Zhang,
and George Jackowski
SYNX PHARMA, Toronto, Ontario, Canada
33.32
Functional Proteomic Platform for
Mapping Cardiac Subproteomes in the
Mouse: A Strategy for the Human Cardiac
Proteome Project
Thomas M. Vondriska, Guang-Wu Wang, Jun Zhang, Xin Joe Qiao, Ernest M. Cardwell, and Peipei Ping
University of California, Los Angeles Department of Physiology, Los Angeles, California 90095
Significant challenges surround large-scale analysis of the human myo-cardial proteome, including: limited availability of tissues, absence of tightly controlled experimental groups, and lack of a concerted strategy to deconstruct protein networks underling cellular function. Thus, we pro-pose an approach to map the human myocardial proteome based on the use of a murine model system. Furthermore, we hypothesize that the mammalian cardiac proteome is organized on the basis of multiple inte-grated subproteomes. We have developed a functional proteomic plat-form that interfaces proteomic techniques (e.g., electrophoresis/mass spectrometry) with biochemical and physiological assays to study how proteins and protein interactions engender phenotype. This platform char-acterizes a subproteome on four levels: 1) protein-profiling (networking proteins in a signaling system); 2) spatial-profiling (role of subcellular localization); 3) temporal-profiling (protein alterations over time following a stimulus); and 4) functional-profiling (changes in protein function associ-ated with altered phenotype). As an example, we have validassoci-ated this platform by characterizing the PKC⑀ signaling subproteome in the heart (however, the platform is applicable across cell type). PKC⑀ (a known cardiac protective protein) was found to form multiprotein complexes with ⬃93 proteins. Assembly of these complexes appears to alter protein localization and affect protein function. Importantly, the physiological phe-notype of cardiac protection appears to be directly linked to modulation of the PKC⑀ subproteome. Integration of studies examining other subpro-teomes will provide a more complete blueprint of cardiac cell function. Importantly, knowledge gained from these mouse studies may be ex-ploited to render analyses of the human cardiac proteome more focused and attainable.
35.1
Proteomics in Biomarker Discovery
Steven A. Carr
Millennium Pharmaceuticals, 640 Memorial Drive, Cambridge, Massachusetts 02139
Proteomics, the quantitative study of the protein content of a cell in a specific state, is a promising new field of scientific endeavor fueled by high resolution separation methods, mass spectrometry and informatics. Pro-gress is being made in the application of proteomics to biologically rele-vant problems, but significant technological challenges remain that are akin to the hurdles faced by the Human Genome initiative a decade ago. In this talk I will discuss some of these challenges, and describe how proteomics is contributing to the drug discovery and development proc-ess at Millennium Pharmaceuticals. I will concentrate on the roles that proteomics is playing in the discovery and validation of biomarkers of disease and pharmacodynamic markers of drug efficacy and mechanism of action. The impact of some recent advances in separation modalities, mass spectrometry and informatics will be highlighted.
35.2
Selecting Targets for Therapeutic
Validation Through Differential Protein
Expression Using Chromatography-Mass
Spectrometry
S. D. Patterson, T. E. Ryan, M. D. Bond, B. M. Domon, and I. N. McCaffery
Celera Genomics Group, Rockville, Maryland
