HUPO First World Congress, November 21–24, Versailles, France 12.6 13.1 Sample Preparation for MALDI-TOF/TOF Protein Identification with >95% Success Rates Proteomic Map of the Human Cholangiocarcinoma Cell Line D. Suckau1, Peter Berndt2, Martin Schuerenberg1, and H. Langen2 1 Bruker Daltonics, 28359 Bremen, Germany; and 2HoffmannLa Roche, 4070 Basel, Switzerland The full characterization of human or bacterial proteomes is a task, which is pursued in industrial proteomics today at increasing pace. As a rough estimate, 50,000 MALDI fingerprint spectra of 2D gel spots are currently required to obtain a basic insight into a proteome. Unfortunately only about 60% of all spectra typically provide for protein identification. Therefore, there is dramatic demand for increased throughput AND success rates of protein identification for industrial proteomics. Here we present technologies, which provide significant improvements of these aspects. Basically, advanced sample preparation protocols and MALDI-TOF/TOF technology allow these performance increases. A novel sample preparation using “AnchorChips” i.e., MALDI sample plates with hydrophobic profiles, was developed subsequently to the automatic digestion of protein spots from 2D gels. The digestion and sample preparation process were fully automated for high throughput analysis. The process does not involve any other purification step, such as microcolumns, etc. AnchorChips with a diameter of 600 m of the hydrophilic sample patch were used, which allow for multiple sample handling steps such as rinsing and recrystallization on the target without increasing the size of the prepared sample patch. Investigated samples came from gels from human embryonic kidney cell lysate. As a first step protein mass fingerprints were recorded with a success rate of 90%. As a second step LIFT-TOF/TOF MS/MS spectra were obtained on a novel MALDI-TOF/TOF mass spectrometer, which increase the rate of protein identification beyond 95%. 12.7 A Transgenic Model for Muscle Gene Expression Profiling In Vivo T. Crepaldi1, P. Accornero1, C. Prunotto1, R. Taulli1, R. Chiarle2, and C. Ponzetto1 1 Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita` di Torino, C.so M. D’Azeglio 52, 10126 Torino, Italy; and 2 Dipartimento di Scienze Biomediche e Oncologia, Universita` di Torino, Via Santena 19, 10126 Torino, Italy The HGF/SF ligand and its receptor (c-Met) promote migration of hypaxial muscle precursors from the somites to their final destination into the limbs, diaphragm and tip of the tongue. They also enhance proliferation of foetal myoblasts and trigger the activation of satellite cells. Activation of Met signaling in cultures of C2C12 cells results in expansion of cycling progenitors and block of myogenic differentiation. To verify the role of Met receptor in muscle differentiation in vivo we produced double transgenic mice where the oncogenic form of Met receptor, Tpr-Met, can be expressed in muscle under the control of tetO responsive promoter. The tTA transactivator was controlled by the Muscle Creatine Kinase (MCK) promoter (Tet-Off system), which is expressed in mouse embryos from E14 dpc onwards, when secondary myogenesis and terminal differentiation occur. In these mice EGFP was used as reporter gene. Five different lines of Tpr-Met transgenics were crossed with homozygous MCK mice. Double transgenics (MCK-TprMet) from only two out of five lines expressed EGFP in muscle. The line with stronger EGFP expression was lethal perinatally. A severe reduction in all skeletal muscles was observed. Most fibres were disorganized and Z-lines were absent. In these animals myotubes did not form and mononucleate cells accumulated. This suggests that Met signaling interferes with muscle terminal differentiation. Total RNA from muscle tissue of these mice will be used as target in microarray experiments versus control RNA from normal muscles. This will allow to delineate an in vivo expression profiling of genes involved in skeletal muscle differentiation. 668 Molecular & Cellular Proteomics 1.9 C. Srisomsap1, P. Subhasitanont1, T. Panichakul1, K. Lirdprapamongkol1, S. Keeratichamroen1, P. Sawangareetrakul1, D. Chokchaichamnankit1, J. Jai-nhuknan2, and S. Sirisinha1,3 1 Chulabhorn Research Institute, Bangkok 10210, Thailand; 2Bruker South-East Asia, Bangkok, Thailand; and 3Mahidol University, Bangkok 10400, Thailand Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct epithelium, occurs with a higher incidence in tropical countries especially in some areas of Southeast Asian countries such as Thailand. Twodimensional (2-D) protein map of human bile duct epithelial carcinoma cell line (HuCCA-1) was studied in parallel with human breast epithelial cancer cell lines (MCF-7) and hepatocellular carcinoma cell lines (HepG2 and HCC-S102). The proteins from MCF-7 were identified by mass spectrometry (MALDI-TOF-MS and ESI/MS/MS) and protein sequencer. Our results show that HuCCA-1 expressed a unique pattern of proteins. Twenty-eight major proteins were identified by matching to the map of MCF-7. We found higher expression of cytokeratin 18 (CK18) but lower expression of glucose regulated protein 75 and heat shock protein 70 in HuCCA-1 compared to MCF-7. CK18 was also expressed at lower level in HepG2 and HCC-S102. There were 2 specific proteins expressed as a series of isoelectric variants, which had MW/pI at 46.2/5.91 and 42.0/5.91 in HuCCA-1, but showed MW/pI at 46.2/5.91 and 42.2/5.99 in HCC-S102. These 2 spots were not expressed in MCF-7 and HepG2 and were identified by ESI/MS/MS as Keratin, type II cytoskeletal 1 (Cytokeratin 1, K1, CK 1, 67 kDa cytokeratin). This 67 kDa intermediate filament protein is normally expressed only in the upper levels of the epidermis but has also found in the serum of papillary thyroid carcinoma as a 35 kDa fragment and in the tissue of esophageal cancer as 67 kDa CK1. Supported by the Chulabhorn Research Institute. HUPO First World Congress, November 21–24, Versailles, France 13.2 13.3 Proteomics of Breast Cancer for Signal Pathway Profiling and Therapeutic Target Discovery Comparative Proteome Analysis of Human Colorectal Cancer Tissues Ikram El Yazidi-Belkoura1, Eric Adriaenssens1, Se´ verine Lottin1, Jean Jacques Curgy1, Anne-Sophie Vercoutter-Edouart2, Je´ roˆ me Lemoine2, and Hubert Hondermarck1 1 EA-1033, Universite´ des Sciences et Technologies de Lille, France; and 2UMR8576, Universite´ des Sciences et Technologies de Lille, France Breast cancer is a major problem for public health, and the identification of new markers as well as the definition of new therapeutic targets, are of critical importance. Practical consequences for treatment derived from a better understanding of the molecular basis of breast cancer cell growth are now emerging, as evidenced by the development of therapeutic strategies based on the inhibition of tyrosine kinase receptors. In this context, methods in functional proteomics have become a powerful approach for deciphering the complex signaling circuitry involved in tumor growth. This is well illustrated with intracellular signaling of fibroblast growth factor-2 (FGF-2) and nerve growth factor (NGF), two potent activators of breast cancer cell survival, proliferation and metastasis (1). We have shown that breast cancer cell transfection with 14-3-3 sigma, a molecular chaperone that is down-regulated in breast cancer cells (2), resulted in a the inhibition of FGF-2 and NGF intracellular signaling. After immunoprecipitation, 2Delectrophoresis and MALDI-TOF analysis, we have identified several targets of 14 –3-3 sigma. Interestingly, HSP90 was also found to interact with PI-3-kinase and IKK and appears to be a crucial element involved in growth signaling as well as a potential therapeutic target as its pharmacological inhibition resulted in breast cancer cell growth arrest and apoptosis. The complementarity between genomics and proteomics for identification of therapeutic targets is well illustrated with H19, an untranslated mRNA, which is oncogenic for breast epithelial cells. H19 transfected breast epithelial cells appear to grow faster in tissue culture, although no molecular target has been assigned to H19 and its mechanism of action remains unknown. Proteomic analysis of H19 transfected cells reveals several modifications of protein synthesis and particularly a strong upregulation of thioredoxin, one of the major proteins regulating intracellular redox metabolism, providing the first molecular target identified for the oncogen H19 (3). Interestingly, this example point out that proteomics can provide usefull data for the understanding of genes and mRNA function, specially in a pathological context. Together with genomics, proteomics is now opening a way to define molecular processes involved in breast cancerogenesis and to identify new markers and therapeutic targets. Technological innovations in large scale/high throughput analysis are now ushering in new prospects. 1. Nurcombe V, Smart CE, Chipperfield H, Cool SM, Boilly B, Hondermarck H (2000) J. Biol. Chem. 275, 30009 –18. 2. Vercoutter-Edouart AS, Lemoine J, Le Bourhis X, Hornez L, Boilly B, Nurcombe V, Revillion F, Peyrat JP, Hondermarck H (2001) Cancer Research 61, 76 – 80. 3. Lottin S, Vercoutter-Edouart AS, Adriaenssens E, Czeszak X, Lemoine J, Morad R, Coll J, Hondermarck H, Dugimont T, and Curgy JJ (2002) Oncogene 21, 1625–1631. Gyong-Sik Ha1, Hyung-Gon Koh1, Kwang-Ho Kim2, Eung-Bum Park2, Wan-Je Park1, Jang-Yun Lee1, and Young-Sun Sohn1 1 R&D Center of Pharmaceuticals, Cheiljedang Corporation, Ichon 467-810, Korea; and 2Mokdong Hospital, Ewha Womans University, Seoul 158-710, Korea Colorectal cancer is one of the most common malignancies in the world. In an attempt to identify proteins that may specifically characterize colorectal cancer, comparative proteome analysis was performed with 5 human cancer tissues and their matching normal tissues, using two-dimensional gel electrophoresis coupled with matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. It was found that 29 protein spots were differentially expressed in at least three tissues, 17 spots significantly increased and 12 spots decreased. Among those protein spots, twelve up-regulated and 2 down-regulated proteins were identified and classified based on their functions by searching SWISS-PROT database. Proteins known to be related to protein folding; protein disulfide isomerase A3 precursor and peptidyl-prolyl cis-trans isomerase A, formation of cytoskeleton; PDZ and LIM domain protein 1 and cofilin, metabolic pathway; alpha enolase and triosephosphate isomerase, gene expression; myc far upstream element-binding protein and heterogeneous nuclear ribonucleoproteins A2/B1, and signal pathway; calgizzarin and putative peroxisomal antioxidant enzyme were overexpressed in cancer tissues, whereas the expression of transgelin related to formation of cytoskeleton and transthyretin precursor to signal pathway decreased. It is expected that these data would contribute to understanding the characteristics of the celluar proteins in colorectal cancer tissues. 13.4 Parallel 2D-DIGE and Microarray Expression Profiling of a Model Breast Cancer Cell System Severine Gharbi, Sarah White, Mariana Bertani, Hong Lin Chan, Michael D. Waterfield, and John F. Timms Ludwig Institute for Cancer Research, University College of London Medical School, London, United Kingdom We report here the use of 2D-Difference gel electrophoresis (DIGE) combined with microarray analysis for differential expression profiling of a breast cancer cell model of ErbB-2 over-expression. The protein expression pattern of a normal human mammary luminal epithelial cell line (HB4a) and a transfected derivative over-expressing ErbB-2 (C3.6) was analysed by 2D-DIGE. Both cell lines were compared at various time points after stimulation with a specific growth factor, heregulin b1 (Hrgb1). As well as increased reproducibility by avoiding gel-to-gel variation, this approach allowed quantitative and statistical evaluation of differential display. Decyder software was used for gel image processing and statistical analysis revealed significant changes between both cell lines in response to Hrgb1. Of 135 features displaying difference in abundance, 27 were identified by in-gel tryptic digestion of spots followed by peptide mass mapping by MALDI/MS. Protein identities were subsequently validated by 1D-western blotting with specific antibodies. A parallel microarray experiment was carried out to evaluate the mRNA levels of 9932 genes between the two cell types under identical growth conditions. A comparison between expression levels of mRNA and the proteins identified herein revealed a good correlation, and a linear regression of 0.66. This indicates post-transcriptional modulation of protein levels. This work is providing us with key information on the regulation of particular proteins in an ErbB2 over expressing cellular system. Molecular & Cellular Proteomics 1.9 669 HUPO First World Congress, November 21–24, Versailles, France 13.5 13.6 Proteomics-based Identification of PGP9.5 Protein Profiling of the Human Epidermis from the Elderly Reveals Up Regulation of a Signature of IFN-␥ Induced Polypeptides That Includes MnSOD and PI3K Myeong J. Nam, Cecilia E. Schmalbach, David E. Misek, Hong Wang, Rork Kuick, and Samir M. Hanash Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109 The detection of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis, as well as providing leads for therapy. We have implemented a proteomic approach to identify proteins that commonly induce an antibody response in colon cancer. Aliquots of proteins from a colon adenocarcinoma cell line (LOVO) were utilized for two-dimensional polyacrylamide gel electrophoresis (2-DE), followed by Western blot analysis in which individual sera were tested for autoantibodies. Sera from 25 newly diagnosed patients with colon cancer, 24 patients with lung adenocarcinoma and 45 non-cancer controls were analyzed. Autoantibodies against a protein identified by Q-TOF mass spectrometry as an isoform of PGP9.5 (ubiquitin C-terminal hydrolase) were detected in 10 of 25 colon cancer patients. They were not detected in 25 sera obtained from normal individuals and patients with colon adenomas. Sera from patients and with lung adenocarcinoma exhibited reactivity against some forms of PGP9.5 but not others. These findings were confirmed by Western blot analysis of both colon tumors and the colon tumor cell line, resolved by 2-DE, using an antibodies to PGP9.5. Expression analysis of microarray data obtained for a panel of 287 different tumors and normal tissues allowed us to correlate serum reactivity with expression patterns at the RNA level. Data based on microarray analysis were confirmed by both RT-PCR and real time PCR. Thus, the findings of anti-PGP9.5 autoantibodies in the serum of patients with colon cancer suggest that this antigen may have utility in colon cancer screening and diagnosis. 670 Molecular & Cellular Proteomics 1.9 Pavel Gromov1, Gunhild Skovgaard2, Hildur Palsdottir3, Irina Gromova1, Morten Ostergaard4, and Julio Celis1 1 Institute of Cancer Biology, The Danish Cancer Society and Danish Centre for Molecular Gerontology, DK-2100 Copenhagen, Denmark; 2 Department of Dermatology, Bispebjerg Hospital, The University of Copenhagen and Danish Centre for Molecular Gerontology, DK-2400 Copenhagen NV, Denmark; 3Max-Planck Institute of Biophysics, 60528 Frankfurt am Main, Germany; and 4Department of Medical Biochemistry and Danish Centre for Molecular Gerontology, The University of Aarhus, DK-8000 Aarhus C, Denmark Aging of the human skin is a complex process that consists of chronological and extrinsic aging; the latter caused mainly by exposure to ultraviolet radiation (photoaging). We have used proteomic profiling technologies and 2D PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. One to two mm3 epidermal biopsies obtained from the forearm of young and old donors were labeled with [35S]methionine for 18 h and subjected to 2D PAGE and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D-gel image of human keratinocytes (http://proteomics.cancer.dk). Quantitative analysis of 172 well-focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, Manganese superoxide dismutase, tryptophanyl tRNA synthetase, phosphatidylinositol 3-kinase, and proteasomal proteins PA 28-␣ and SSP 0107), that is induced by IFN-␥ in primary human keratinocytes, and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Besides arguing for a role of IFN-␥ in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo. HUPO First World Congress, November 21–24, Versailles, France 13.7 13.9 Two-dimensional Molecular Profiling of Mantle Cell Lymphoma Human Liver Tissue Two-dimensional Gel Electrophoresis Map and Hepatocellular Carcinoma-related Proteins Francesca Antonucci1, Marco Chilosi2, Claudia Parolini2, Mahmoud Hamdan3, Hubert Astner3, and Pier Giorgio Righetti1 1 University of Verona, Department of Agricultural and Industrial Biotechnologies, Verona, 37134, Italy; 2University of Verona, Department of Pathology, Verona, 37134, Italy; and 3Computational, Analytical & Structural Sciences, GlaxoSmithKline, Verona, 37135, Italy In present study we have compared 2-D maps of reactive lymph-nodes and Mantle Cell Lymphoma tissue. Mass-spectrometry-compatible-colloidal Coomassie has revealed a total of ca. 750 spots in each maps. Comparison of the 2-D maps by PDQuest established up- and downregulation of ca. 150 spots, with positive variations (up to 10 folds) and negative variations (up to 13 folds). More than 20 proteins have been identified by MALDI-TOF mass spectrometry, with an additional five spots which could not be matched to any of the available databases. Some proteins, such as the 78 kDa glucose-regulated protein precursor, appear to be in common with other tumours. Others reflect changes in cellular metabolism. T-cell leukemia/lymphoma protein 1A over-expression agrees with the MCL microenvironment. Jina Kim, Na-Young Ha, Jin Sook Ahn, Seung Uook Lee, Yu Na Kim, Bok Im Cho, Sung-Jo Kang, Chang-Won Lee, and Jae Won Kim Division of Life Science, Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, South Korea Hepatocellular carcinoma is a common malignancy worldwide and is a leading cause of death. In recent years, a proteome analysis technology is one of the most popular tools approaching cancer studies. The aim of this work was to largely expand the currently available human liver tissue map and to study the differential expression of proteins in tumor and normal tissues. Proteins were separated in the first dimension by isoelectric focusing on IPG strips and by 7.5–17.5% gradient SDS-PAGE gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight mass spectrometry (DE-MALDI-TOF MS). Up to the present, reference 2-DE maps of human liver tumor tissue include 212 protein spots (117 spots in pH 4 –7 map and 95 spots in pH 6 –9) corresponding to 127 different polypeptide chains. And we analyzed the differential protein expression of liver tumor samples. Proteins whose expression levels were different by more than three fold in at least 30% (four) of the 11 patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, one overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. 13.8 13.10 Proteome Analysis of Gastric Cancer: Overexpression of Thymidine Phosphorylase in Gastric Cancer Tissue Proteome Analysis of Gastric Cancer: Overexpression of Rho GDP Dissociation Inhibitor 1 in Human Gastric Cancer Tissues Na-Young Ha, Jina Kim, Jin Sook Ahn, Bok Im Cho, Yu Na Kim, Seung Uook Lee, Sung-Jo Kang, Jae Won Kim, and Chang-Won Lee Division of Life Science, Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, South Korea Gastric cancer is the second most common cause of cancer-related mortality worldwide and the 14th overall cause of death. Proteomic approach was undertaken for the analysis of differential expression of protein between stomach cancer tissue and adjacent non-cancerous region. So it is a powerful tool to find out candidate proteins for diagnostic markers and therapeutic targets. Protein samples were prepared from cancer and normal tissues of the same patients, and were subjected to isoelectric focusing on immobilized pH 4 –7 gradient strips followed by 7.5–17.5% gradient SDS-PAGE gels. Protein spots were detected by staining with silver nitrate. Image analysis was carried out with PDQuest software. Paired samples of seventy gastric cancer patients were obtained from Gyeongsang National University Hospital and Inha University Hospital and were analyzed by two-dimensional gel electrophoresis. Of the seventy samples that gave readable gel images thirty five samples were positive for overexpression (with a cutoff value of 3.0) of the spot NO.4528. The spot was excised from preparative gels, digested by trypsin, and subjected to peptide mass fingerprinting. The spot was identified to be thymidine phosphorylase, with 16 matching peptides which corresponds to a sequence coverage of 34%. Yu Na Kim, Bok Im Cho, Na-Young Ha, Jina Kim, Jin Sook Ahn, Seung Uook Lee, Sung-Jo Kang, Jae Won Kim, and Chang-Won Lee Division of Life Science, Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, South Korea Gastric cancer remains a leading cause of cancer-related death worldwide. Advances in diagnostic and treatment technologies have enabled us to offer excellent long-term survival results for early gastric cancer, but prognosis of advanced gastric cancer still remains poor. The aim of this work was to figure out the protein components that are differentially expressed in stomach cancer tissues to develop the protein candidates for tumor marker. In this study, we have analyzed the proteomes of 70 stomach cancer tissue samples and compared with those of noncancerous tissues of the same patients. Human stomach tissue samples were prepared from resection materials of gastric cancer patients. The proteins of stomach tissues were analyzed by two-dimensional electrophoresis, stained with silver nitrate and the images were analyzed with the aid of PDQuest. Of the 70 samples that gave readable gel image, 4 samples were positive for overexpression (with a cutoff value of 3.0) of the spot no. 3211. The spot was excised from preparative gels, digested by trypsin, and subjected to peptide mass fingerprinting. The spot was identified to be Rho GDP dissociation inhibitor 1 (Rho GDI 1), with the sequence coverage of 28%. The Rho GDI forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. Rho GDI has been implicated in tumor cell apoptosis, invasion and metastases. In the previous studies reported, overexpression of Rho GDI has been associated with chemoresistance in several cancer cell lines. Molecular & Cellular Proteomics 1.9 671 HUPO First World Congress, November 21–24, Versailles, France 13.11 13.13 Proteome Analysis of Gastric Cancer: Overexpression of Galectin-1 in Human Gastric Cancer Tissue Proteomic Analysis of Tyrosine Phosphorylated Proteins in Human MCF10A Mammary Epithelial Cells Treated by HGF/SF Bok Im Cho, Yu Na Kim, Na-Young Ha, Jina Kim, Jin Sook Ahn, Seung Uook Lee, Sung-Jo Kang, Jae Won Kim, and Chang-Won Lee Division of Life Science, Research Institute of Life Sciences, Gyeongsang National Universit, Jinju 660-701, South Korea Gastric cancer remains a great challenge for clinicians and scientists. It is one of the most frequent cancers worldwide, and it is the second most common cause of cancer-related deaths. Patients with gastric cancers have a poor prognosis and low survival rates. The aim of this work was to figure out of the protein components that are differentially expressed in gastric cancer. In this study, we analyzed proteomes of gastric cancer tissue samples and adjacent noncancerous tissues of same patients. The protein of gastric cancer and normal tissue were separated two-dimensional gel electrophoresis, and stained with silver nitrate. The images of stained gels were analyzed with aids software, PDQuest. Paired samples were obtained from 70 gastric cancer patients in Gyeongsang National University Hospital and Inha University Hospital in South Korea. Of the 70 samples that gave readable gel images, 8 samples were positive for overexpression (with a cutoff value of 3.0) of the spot no. 3023. The spot was excised from preparative gels, digested by trypsin, and subjected to peptide mass fingerprinting. The spot was identified to be galectin-1, with 8 matching peptides which corresponds to a sequence coverage of 74%. Galectin-1 with molecular masses of about 14.5 kilodaltons have been found in a variety of normal and malignant cells and have been implicated in the regulation of cell growth, cell adhesion, and metastasis. 13.12 A New Look at the Tumor Proteome by Large-scale Analysis of MHC Peptides Arie Admon1, Eilon Barnea2, Ilan Beer2, Lior Dassau1, and Tamar Ziv1 1 Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel; and 2IBM Research Laboratory, Haifa, Israel Among the thousands of proteins expressed in cells, there are many that do not accumulate to a significant level due to their rapid degradation and therefore remain undetectable by standard proteomics approaches. Some of the peptides resulting from these degradations end up being stabilized and so accumulate while displayed within the Major Histocompatibility Complex (MHC) molecules on the cell surface. The amounts of these peptides correlate with the protein degradation rates and with their binding affinity to the MHC molecules. In Addition to peptides derived from normal cellular proteins, cancer cells display peptides originating from Tumor Associated Antigens, which attract significant attention as candidates for development of cancer vaccines. We describe here the construction of large datasets of MHC peptides presented by various human cancer cell types. The cells were transfected with vectors for secreted, soluble MHC molecules (sMHC), the sMHC were recovered in large amounts from the growth medium and the bound peptides were analyzed by capillary ESI-LC-MS/MS. Peptides were identified from human lung, breast, ovarian, and prostate tumor cells, from the MHC haplotypes: A2, B7 and Cw4. As expected, the majority of the peptides were derived from housekeeping proteins and only a few originated from tumor antigens or from yet-uncharacterized proteins. A catalogue of these peptides is available online with relevant links. A new bioinformatics tool, Pep-Miner, described in a separate poster, vastly facilitated the analysis. The approach described here offers a staging point for a ‘human cancer MHC-peptides project’, to follow cancer genomics and proteomics analyses. 672 Molecular & Cellular Proteomics 1.9 Julien Deheuninck, Sylvie Reveneau, David Tulasne, Catherine Leroy, Bernard Vandenbunder, Yvan de Launoit, and Ve´ ronique Fafeur CNRS UMR8117, Institut de biologie de Lille, IPL, 1, rue du Pr Calmette, 59021 Lille, France Hepatocyte growth factor/scatter factor (HGF/SF) induces epithelial cell proliferation, scattering and morphogenesis through activation of the MET tyrosine kinase receptor. As deregulation of HGF/SF signalling is involved in tumoral and metastatic progression, our goal is to improve the knowledge of molecular actors of cancer, in particular for breast cancer. According to numerous studies in different cell types, HGF/SF regulates by phosphorylation an extensive number of proteins. To clarify their relevance and to possibly discover novel targets of HGF/SF signalling, we decided to investigate exhaustively tyrosine phosphorylated proteins in a single human mammary epithelial cell line, in which HGF/SF is biologically active. We first compared HGF/SF activity in human mammary epithelial cell lines (MCF-7, MDA-MB231, MCF-10A). This led us to further investigate MCF-10A cells, which : 1) express mammary specific epithelial markers, 2) are sensitive to HGF/SF treatment, with efficient phosphorylation of known targets such as MET, GAB1, ERK or AKT and 3) are biologically responsive to HGF/SF, with efficient cell scattering at low cell density or in wounding experiments. To identify phosphoproteins regulated by HGF/ SF, we first performed immunoprecipitation/immunoblotting experiments, using anti-phosphotyrosine antibodies. Following separation by monodimensional electrophoresis, 4 bands were induced after HGF/SF treatment (of ⬃180, 165, 120 and 75 kDa). These proteins are not identified and do not correspond to GAB1, CBL, nor SHC. Further experiments are underway for their identification by mass spectrometry. Alternatively, we are using two-dimensional electrophoresis of whole protein extract followed by immunoblotting using anti-phosphotyrosine antibodies and final identification by mass spectrometry. HUPO First World Congress, November 21–24, Versailles, France 13.14 13.16 Database for Fluorescence 2D Difference Gel Electrophoresis (2D-DIGE) Protein Expression Fingerprints of Normal Segments of the Colon Yasuharu Mori, Tadashi Kondo, Tesshi Yamada, and Setsuo Hirohashi Irina Gromova, Pavel Gromov, and Julio Celis Cancer Proteomics Project, National Cancer Center Research Institute Fluorescence 2D Difference Gel Electrophoresis (2D-DIGE) has been used for several biological studies. In 2D-DIGE, protein lysates are labeled with different fluorescence dyes and separated concomitantly in a same gel. Because samples can be run with a common control sample in a same gel, accurate spot matching can be performed with broad dynamic range of fluorescence signals. We aimed to construct 2D database and to evaluate a potential of 2D-DIGE for oncological studies. Protein extracted from a human colon cancer cell line, DLD-1, was labeled with Cy5 and separated according to their isoelectric point and molecular weight. Among 2,000 spots observed, approximately 500 spots were chosen randomly and 343 spots were identified using mass spectrometry and database search. They were grouped based on their molecular function according to Panther Categories in Celera Discovery System. Majorities of identified proteins were a family of nucleic acid binding protein, oxidoreductases, cytoskeletal proteins and chaperons. Although one receptor and six proteins for signal transduction were observed, proteins with lower amount, such as transcription factors and cell cycle regulators, were not observed. These results indicated that, 2D-DIGE has a limited potential to access mechanisms of cancer biology. Biopsied tissues of human colon cancer were also subjected to 2D after they were labeled with Cy5. Most of identified spots in 2D database of DLD-1 cells were observed in 2D images of human colon cancer tissues. These results suggest that the database for 2D-DIGE using DLD-1 cells will be useful for rapid spot identification of clinical samples. Institute of Cancer Biology and Danish Center for Human Genome Research Colorectal cancer is one of the most common types of cancers in developed countries. Despite advances in treatment this disease remains life threatening for a large number of people. Today, about 50% of all patients with colorectal cancer are diagnosed with early stage cancer, and most of these patients will be cured by surgery. However, approximately 30% of these patients will experience recurrence over the next 5 years. Clearly, survival can be improved if patients are diagnosed at early stages of the disease. In our laboratory we are using proteomic technologies to compare the protein expression of normal and cancerous fresh colon biopsies with the aim of revealing molecular signatures that may identify lesions at a very early stage of the disease. One of the major challenges we face with these studies relates to the fact that various segments of the normal colon epithelia have different susceptibilities to neoplastic transformation and may therefore express distinct proteome profiles. Here we present initial studies intended to characterize the proteome profiles of normal colon biopsies obtained from various regions of the colon. 13.15 13.17 Clustering Analysis of Human Cancer Cell Lines Based on Quantitative 2D Mass Spectrometry-based Analysis of Tubulin Isoforms in Taxol-sensitive and -resistant Human Cancer Cell Lines Masahiro Seike, Tadashi Kondo, Tesshi Yamada, and Setsuo Hirohashi Cancer Proteomics Project, National Cancer Center Research Institute We aimed to perform clustering analysis of human cancer cell lines using fluorescence 2D Difference Gel Electrophoresis (2D-DIGE). In 2D-DIGE, different samples are labeled with different fluorescence dyes and coseparated in a same gel. Because samples are run with a common sample in a same gel, accurate and rapid spot matching can be performed with broad and quantitative dynamic range of fluorescence signals. To evaluate a potential of 2D-DIGE for clustering analysis, two models of human cancer cell lines were studied. First, cell lines of the same histological type from different organs were examined. Adenocarcinoma cell lines derived from lung, pancreas or colon cancer, were analysed by 2D-DIGE. Each group involved 10 cell lines. A set of quantitative intensity of 180 spots was subjected to clustering analysis. Clustering analysis resulted that most cell lines used were grouped into their original organ. The proteins used to segregate each group from the other ones included nucleoside diphosphate kinases. Second, cell lines of different histological types in a same organ were examined. Thirty lung cancer cell lines of adenocarcinoma, squamous cell and small cell cancer were subjected to clustering analysis using quantitative data of 170 spots as above. The cell lines were grouped into their original histological types using spots including stathmin and keratin19. These results indicate that 2D-DIGE is a powerful tool for clustering analysis of human cell lines and suggest possible applications for clinical uses. Pascal Verdier-Pinard, Fang Wang, Berta Burd, Susan Horwitz, and George Orr Albert Einstein College of Medicine, Bronx, New York 10461 Alterations to microtubule stability may be a crucial determinant in the development of clinical resistance towards Taxol and other drugs with a binding site on the microtubule polymer. Cancer cells may alter their microtubule dynamics by differential expression of the six ␣-tubulin and seven -tubulin isotypes, by mutation of tubulin, or by differential posttranslational modifications of these tubulin isotypes. Most of the primary sequence divergence in the various tubulin isotypes and all of the posttranslational modifications, except acetylation, occur within the C-terminal 20 amino acids. We have developed mass spectrometry-based methods for the analysis of tubulin isoforms from human cancer cell lines. In the cell lines examined, the major tubulin isotypes were K␣1 and I, the next abundant tubulin isotypes were ␣6 and IVb, and the minor tubulin isotypes were ␣4 and III. Only trace amounts of monoglutamylated tubulins were detected and ␣ tubulins were almost totally tyrosinated. Additionally, we detected and quantified the expression of mutant tubulin in Taxol- and epothilone-resistant cell lines. These studies represent the first comprehensive analysis of tubulin isoform expression at the protein level in cancer cells. We seek to adapt these approaches to normal and malignant tissues, and to design methodologies for absolute quantification of tubulin isoforms in human samples. Molecular & Cellular Proteomics 1.9 673 HUPO First World Congress, November 21–24, Versailles, France 13.18 13.20 Proteomic Studies on Hepatocellular Carcinoma Cell Surface Expression of Heat Shock Proteins in Human Leukemia Cell Lines Qi-chang Xia1, Rong Zeng2, Zhao-you Tang3, and Hong-yang Wang4 Jun Ho Jang, Bong Kyung Shin, David E. Misek, and Samir M. Hanash 1 Research Center for Proteome Analysis, Shanghai Inst.of Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai, 200031, China; 2Research Center forProteome Analysis, Shanghai Inst.of Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai, 200031, China; 3Shanghai Zhongshan Hospital, Shanghai, 200032, China; and 4Eastern Hepatobilliary Surgery Institute, Shanghai, 200438, China Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109 We demonstrated the comparative proteomic analysis of human hepatocellular carcinoma (HCC). Firstly, two clones with high (MHCC97-H) and low (MHCC97-L) were isolated from the parent cell line, which have the same genetic background but have different metastatic behaviours. Then, the proteomic profiles of the two cell clones were established and compared based on two dimensional electrophoresis. Several proteins with different expression levels were further identified by MALDI-TOF-MS characterization, indicating their relation with the HCC metastatic mechanism. Furthermore, immuno-blotting analysis on over 60 clinical tissues of hepatocellular carcinoma also confirmed the observations on cultured cells. In addition, directed comparisons of clinical tumor and normal tissues were carried out to find new markers for the carcinogenesis of HCC. Our analysis gave insight into the carcinogenesis and metastatic mechanism and helped us to find out clinical markers of hepatocellular carcinoma. The plasma membrane is a sub-cellular compartment of substantial interest in various aspects of cancer, from molecular diagnosis to cancer cell immune system avoidance. Profiling of the cell surface proteome in both normal and cancer cells provides an effective approach for the identification of novel targets for diagnostics and therapeutics. We have implemented a biotinylation-based strategy for targeting plasma membranederived proteins to identify cell surface proteins of leukemia cell lines. Following biotinylation of the Sup-B15 (human acute lymphoblastic leukemia B cell line) and the U 937 (human acute monoblastic leukemia cell line, the labeled proteins were affinity-captured and purified on streptavidin columns, further resolved by 2-D PAGE, then transferred to PVDF membranes. The biotinylated proteins were visualized by hybridization with streptavidin/Horseradish peroxidase complex, then identified from silver-stained gels using MALDI and tandem mass spectrometry. Remarkably, a large set of heat-shock proteins, including GRP78, GRP 75, heat shock 70 kDa protein 2, heat shock 70 kDa protein 9B (mortilin-2), hsp72, heat shock 60 kDa protein 1 and heat shock 27 kDa protein, all previously considered to be associated with the endoplasmic reticulum were found to be highly abundant on the cell surface. The findings were correlated with gene expression at the RNA level by DNA microarray analysis. Further investigation of heat shock proteins, thought to be chaperones in antigen presentation, may help elucidate immune system avoidance mechanisms of human leukemia cells. 13.19 13.21 New Differentially Expressed Stomach Cancer Markers Identified through Extended Proteomics Analysis on Highly Selected Tumor Samples Proteome Analysis of Gastric Cancer: Characterization of Proteome Analysis Data and Comparison of Pre-processing Methods for the Differential Expression Analysis M. L. Fogeron1* S. Lamer1*, S. Heim1, Ch. Ro¨ cken2, M. Ebert2, and P. von Hoegen1 1 2 Europroteome, 16761 Henningsdorf/Berlin, Germany; and Otto-von-Guericke University, 39120 Magdeburg, Germany A case study was performed on stomach cancer tumor samples to demonstrate that the unique combination of a collection of highly purified, well documented, clinical specimens with modern proteomics and transcriptomics technologies can lead to the identification of multiple new stomach tumor markers with value for therapy and diagnostics. These protein markers have been identified by leveraging EUROPROTEOME’s multidimensional system biology technology infrastructure (e.g., Proteomics, Transcriptomics and Bioinformatics) and using purified human tissue samples (applying the proprietary sample preparation method) from the company’s extensive human tissue sample bank. Applying 2D gels of normal and tumor tissues over 350 differentially expressed marker spots could be identified. Many of these proteins have been altered in several patients indicating the general relevance of them for the disease. Many known changes in regard of de-differentiation and increased proliferation could be confirmed and thereby validated our approach. After extensive analysis nearly 50 markers could be identified as yet unknown or as new stomach-cancer-associated markers. These provide a significant and proprietary list of new markers with potential for use in diagnostic and therapy of cancer, an example being EP32.1.2. Many of these markers are likely to be drug candidates (e.g., enzymes) making them ideal candidates for therapeutic product development. * Contributed equally. 674 Molecular & Cellular Proteomics 1.9 Jae Won Kim, Sung Jin Ahn, Euy Hoon Suh, Jong Min Bae, and Chang-Won Lee Gyeongsang National University, Jinju 660-701, Korea Gastric cancer is one of the leading causes of death worldwide. The identification of new markers and therapeutic targets is of critical importance, and a proteomics project on gastric cancer has been launched three years ago as an effort to attain the goal. Selection of differentially expressed proteins in tumor and normal tissues would be the first rational step toward finding markers and targets of the disease. Two-dimensional gel electrophoresis was used as the main technological platform of proteome analysis, and proteome data for matched tissue samples from forty gastric cancer patients have been generated by employing immobilized pH gradient (pH 4 –7) isoelectric focusing in the first dimension and 7.5– 17.5% gradient SDS-PAGE in the second dimension. Protein visualization was done by staining with silver nitrate. The images were transformed into digital data by scanning and analyzed by PDQuest software for the automatic spot detection. As a prerequisite to the analysis of proteome data, it is important to have a deep understanding the characteristics of the data, which will enable to select the most appropriate analysis method(s) that take the data characteristics best into consideration. Here, we have characterized the proteome data obtained from the analysis of clinical tissues, with respect to the spot numbers, spot intensities, and reproducibilities. Also, three different normalization methods were used to preprocess the raw data, and the t-test was performed to select the differentially expressed proteins. The results are compared and discussed. HUPO First World Congress, November 21–24, Versailles, France 13.22 13.24 Proteomics-based Microarray Technology for the Identification of Tumor Antigens Differential Expression of Sub-proteome Between Human Liver Cancer HepG2 and Hep3B Cell Lines Juan Madoz-Gurpide, David E. Misek, Hong Wang, and Samir M. Hanash Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109 The identification of circulating tumor antigens that elicit a humoral response provides a means for early cancer diagnosis. We have developed a novel proteomic method for probing the cell and tissue proteome by combining liquid-phase protein separations with microarray technology. The advantage of a liquid-based separation system is that proteins in hundreds of individual fractions can be spotted onto microarrays directly and used as targets for a variety of probes. These microarrays were probed with the sera of cancer patients in order to detect disease-related autoantibodies. The A549 lung adenocarcinoma cell line was resolved by isoelectric focusing (Rotofor) in the first dimension. Each of twenty fractions was further separated by RP-HPLC, such that 88 fractions were obtained for each first-dimension fraction. All 1760 fractions and controls were arrayed in duplicate onto surface-modified glass slides. An antibody against a previously identified cancer biomarker was used to detect 15 specific antigen-containing fractions (p ⬍ 0.01 and ratio ⬎ 2). The presence of the antigen in these fractions was confirmed by Q-TOF mass spectrometry. The microarrays were individually hybridized with sera from 12 colon cancer patients, from 12 lung cancer patients as well as from 12 normal individuals. The reactivity of 15 selected fractions was analyzed. 8/12 colon patient sera and 10/12 lung cancer sera reacted positively, whereas only 1/12 normal controls showed reactivity. The proteomic approach we have developed has utility for the development of serumbased assays for cancer screening and diagnosis. Chi-Yue Wu1,2, Szu-Pei Wu1,2, Chih-Lei Lee1, Shiuan-Yi Huang2, Cheng-Liang Chen2, Ying-Ta Wu1, Kay-Hooi Khoo2, Shui-Tein Chen2, and Andrew H.-J. Wang1 1 Core Facilities for Proteomic Research Academia Sinica, Taipei, Taiwan; and 2Institute of Biological Chemistry Academia Sinica, Taipei, Taiwan In the search for new protein markers of human liver cancer, the method of quantitative two-dimensional gel electrophoresis coupled with mass spectrometry was applied to define the protein expression profiles of human liver cancer HepG2 and Hep3B cell lines. Gel image matching and statistic analysis showed that of the total 792 protein spots that were matched, 537 spots were differentially expressed at a significant level. In total, 111 protein spots were identified by peptide mass fingerprinting combined with database searching. These identified proteins were classified into several functional groups, including chaperones and stress response proteins, cytoskeletal proteins, enzymes involved in metabolism and biosynthesis, and proteins with regulatory functions. Among these differentially expressed proteins were several cancer-related proteins, i.e., annexin II, cathepsin B, dihydrodiol dehydrogenase II, heterogeneous nuclear ribonucleoproteins (hnRNP A1, hnRNP A2/B1 and hnRNP C), heat shock proteins (Hsp90 and Hsp70) and glucose-regulated proteins (Grp78 and Grp94). The strategy of rapid comparative proteomic studies of HepG2 and Hep3B cells is demonstrated here to be effective in narrowing down the range for screening novel cancer markers from the sea of whole cell proteins. The experimentally derived model provides important leads and several potential targets for the development of diagnostics and therapeutics for liver cancer, a common cancer in Asia and Africa. 13.23 Comprehensive Profiling of Surface Membrane Proteins of Cancer Cells Bong Kyung Shin, Jun Ho Jang, David E. Misek, Rong Zhao, Hong Wang, Rork Kuick, and Samir M. Hanash Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109 Comprehensive profiling of surface membrane proteins of cancer cells is highly relevant to cancer diagnostics and therapeutics and to our understanding of dysregulated pathways. Biotinylation of surface membrane proteins of intact cells followed by selective capture of biotinylated proteins using avidin columns allowed us to undertake comprehensive profiling of surface membrane proteins of a variety of cancer cell lines and freshly isolated tumor cells. Proteins were identified by MALDI and tandem mass spectrometry analysis and finding were correlated with gene expression at the RNA level by DNA microarray analysis. A lung adenocarcinoma cell line (A549), a colon adenocarcinoma cell line (Lovo), a neuroblastoma cell line (Sy5y), a B-cell lymphoblastic leukemia cell line (Sup-B15), and freshly isolated ovarian tumor cells were analyzed. Distinctive patterns of expression were observed for each cell population based on patterns analysis and the identification of over 100 surface membrane proteins. For example, Annexin III which was present in A549, Lovo and ovarian tumor cells but not in Sy5y or Sup-B15. Other proteins such as heat shock 70 kD protein 2, HLA class I histocompatibility antigen, membrane associated progesterone receptor component 1, and heat shock 27 kD protein 1, showed significant quantitative difference in expression in different cell types both in gel image analysis and RNA measurement. Comprehensive analysis of surface membrane proteins of cancer cells provides an effective approach for the identification of cancer proteins of diagnostic and therapeutic value. Molecular & Cellular Proteomics 1.9 675 HUPO First World Congress, November 21–24, Versailles, France 13.25 13.26 Strategic Experimental Approach Towards Establishing Model Hepatoma Cell Lines for the Global Human Liver Proteome Initiatives Proteomic-based Identification of Tumor Antigens in Pancreatic Cancer Chih-Lei Lee1, Wei-Tzer Shyu1, Chin-Yi Lin1, He-Hsuan Hsiao1, Cheng-Liang Chen2, Chi-Yue Wu1, Ying-Ta Wu2, Kay-Hooi Khoo2, and Andrew H.-J. Wang1,2 Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109 1 Core Facilities for Proteomic Research, Academia Sinica, Taipei, Taiwan; and 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Liver cancer is a world-wide disease which has serious impact in Asia and Africa and has become a leading cause of death in these areas. Recognizing the importance of liver diseases, the Asian and Oceanian Human Proteome Organization (AOHUPO) has launched the human liver proteome project as one of its major initiatives. The well established human hepatoma HepG2 (from hepatitis B surface antigen-negative patient) and Hep3B (from a HBsAg-positive patient) cells are the designated in vitro experimental model cell lines. In response to this initiative and to support the nation-wide genomics and proteomics research programs, a National Core Facilities for Proteomic Research was established in Taiwan a year ago. A variety of proteomics related projects including in depth, comprehensive mapping of the hepatoma proteome were initiated which serve to drive innovative technical developments in sample preparation, separation, identification, and further characterization. Both gel electrophoresisbased and liquid chromatography-based separation systems coupled with integrated high-throughput mass spectrometer systems (including ESI-ion trap, ESI-Q/TOF, MALDI-Q/TOF, and MALDI-TOF/TOF) are currently in routine operation. At present, more than 1000 proteins from the human HepG2 and Hep3B liver cell proteomes have been identified using either or both approaches. The well recognized pitfalls in either methods alone dictate that both platform technologies coupled with well designed fractionation and enrichment strategies are required if a true human proteome database is to be established. Our data are compiled and presented here to serve as an entre´ e for AOHUPO to set the standards and regulations in the global human proteome initiatives. Su-Hyung Hong, David E. Misek, Hong Wang, Rong Zhao, Craig Logsdon, and Samir M. Hanash Pancreatic cancer has a poor prognosis, in part due to lack of early detection. There is substantial interest in the identification of human tumor antigens and autoantibodies for early diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in pancreatic cancer. Proteins from two pancreatic cancer cell lines (Panc-1 and BxPC3) were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for autoantibodies. Sera from 23 newly diagnosed patients with pancreatic cancer, 14 patients with lung adenocarcinoma and 15 healthy subjects were analyzed. Autoantibodies against a novel PDZ-Domain and LIM-Domain protein, identified by MALDI in the BxPC3 cell line were detected in sera from 9 of 23 patients with pancreatic cancer, in 1 of 14 patients with lung cancer but were not detected in sera from normal individuals. In the Panc-1 cell line autoantibodies were detected against a calcium-binding protein (in 11 of 23 pancreatic cancer sera) and against a phosphatase (in 8 of 23 pancreatic cancer sera), but neither was detected in sera from 15 normal individuals. The phosphatase was detected by 1 of 14 patients with lung cancer and the calcium-binding protein was detected in sera from 8 of 14 patients. These tumor-associated antigens may have utility as a biomarker for pancreatic cancer screening and diagnosis. 13.27 Proteome Mapping of Chronic Lymphocytic Leukemia C. A. Evans1, D. A. E Cochran1, D. Blinco1, J. Burthem1, S. K. Stevenson2, S. J. Gaskell1, and A. D. Whetton1 1 Leukaemia Research Fund Proteomics Facility, UMIST, Manchester, United Kindgom; and 2Southampton General Hospital, Southampton, United Kingdom B-Cell Chronic Lymphocytic Leukaemia (B-CLL) can be divided into two groups depending on the mutational status of the VH locus. Ig VH status correlates with prognosis. Cases with somatic mutation Ig VH CLL (M-CLL) have a relatively less aggressive form of CLL compared to patients with unmutated Ig VH CLL (UM-CLL). Despite this, the gene expression profiles of UM-CLL and M-CLL Ig VH are extremely similar. We have undertaken a proteomic analysis of these prognostic groups to identify proteins that may have prognostic value in distinguishing between these clinical subtypes. Differential protein expression profiling was performed using 2D-gel electrophoresis. Similar patterns of protein expression were obtained for all patients. The gels were subjected to computerised image analysis using the Progenesis/Progenera software. Four proteins were identified as present/absent between UM-CLL and M-CLL. Principal Component Analysis confirmed that there were significant differences in the patterns of protein expression analysed by 2-dimensional gel electrophoresis. Over 65 proteins were identified from gels using MALDI-ToF mass spectrometry. One of these was the apoptotic regulator, Smac/DIABLO. It was expressed only in cases where Ig VH was mutated; the less aggressive form of the disease. Proteomic analysis thus has value in determining new prognostic and mechanistic detail in the leukaemias. 676 Molecular & Cellular Proteomics 1.9 HUPO First World Congress, November 21–24, Versailles, France 13.28 13.29 Identification of Metastasis-associated Proteins by Proteomic Analysis and Functional Exploration of IL-18 in Metastasis Using Antibody Microarray for Proteomic Analysis of Esophageal Squamons Cell Carcinoma Daifeng Jiang1,2, Wantao Ying1, Yinglin Lu3, Jinghong Wan1, Yun Zhai1, Wanli Liu1, Zongyin Qiu2, Xiaohong Qian1, and Fuchu He1 1 Beijing Institute of Radiation Medicine, Beijing, 100850, People’s Republic of China; 2Chongqing University of Medical Science, Chongqing, 400000, People’s Republic of China; and 3Institute of Basic Medical Sciences, Beijing, 100850, People’s Republic of China Protein phosphorylation is the most important reversible post-translational modification that occurs in cells. Widespread metastasis is the major lethal cause of cancer. So far, very little is known about its mechanisms. In the present study, comparative proteomic analysis was used to find out metastasis-associated proteome. Firstly, a pair of highly and lowly metastatic sublines (termed as PLA801D and PLA801C respectively), originated from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, their protein expression profiles were compared by a proteomic approach. Of them, 11 metastasis-associated proteins were identified and further validated by 1-D western blotting, northern blot and/or semi-quantitative RT-PCR analysis. Compared with that in lowly metastatic PLA801C subline, CK18, TGLC, GDIR, TPMF, IL-18 and ANX1 were significantly up-regulated, while ER60, CH60, PDX1, CLI1 and KCRB were significantly down-regulated in highly metatsatic PLA801D subline. Intriguingly, all the candidate proteins except CLI1 have been evidenced to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was only present in highly metastatic PLA801D, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense or IL-18 antisense into PLA801C or PLA801D subline respectively. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, and that IL-18 antisense remarkably decreased the invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play role in metastasis by inhibiting E-cadherin expression. Xiaohang Zhao, Yu Liu, Yousheng Mao, Huixin Wang, Xiaoguang Ni, Fang Liu, and Min Wu Cancer Institute of Chinese Academy of Medical Sciences, Beijing 100021, People’s Republic of China Esophageal squamons cell carcinoma (ESCC) has very poor prognosis. The early detection of ESCC is very depends on the discovery of some specific and sensitive molecular biomarkers. We have studied the proteomic alterations of ESCC using antibody microarrays in a comparative fluorescence assay to measure the abundance of many specific cellular proteins simultaneously. Two groups samples, one based on the individual patient’s tissue for comparative quantitation, the other based on the pooled samples for subtraction of the individual differences, were labeled by covalent attachment of spectrally resolvable fluorescent dyes. Specific antibody-antigen interactions localized specific components in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein’s abundance. Several kinds of proteins which low- or highexpressed in both groups involving in different signaling pathways and cell cycle control, such as apoptosis pathway and cell cycle progression. These results suggest that protein microarrays can provide a practical approach to characterize patterns of variation in hundreds of thousands of different proteins in proteomic analysis. This work was supported by grants from the special funds for Major State Basic Research (G19980512), State 863 High-tech R&D (2001AA227091) and the NNSF (39990570 and 30171049) of China. 13.30 Metabolic Proteome Analysis of Hepatocellular Carcinoma Kang-Sik Park1, Hoguen Kim2, and Young-Ki Paik1,3 1 Yonsei Proteome Research Center, Yonsei University; 2Department of Pathology, Yonsei University School of Medicine, Yonsei University; and 3Department of Biochemistry, Yonsei University To understand functional roles of proteins in hepatocelluar carcinoma (HCC) at the protein level, 19 cases of liver tumor tissues were analyzed by proteomic tools. Results were compared with those of paired adjacent nontumorous tissues. We were able to identify more than 150 protein spots out of 1000 spots in HCC in which total 32 types of protein spots representing 24 protein families were induced while 34 types of protein spots representing 27 protein families decreased. These differentially expressed proteins were confirmed by Western blot analysis. To our surprise, most of the induced proteins were closely related to metabolic pathway of ATP synthesis and amino acid transformation. Interestingly, the decreased proteins are liver-specific proteins involved in detoxification. This result is consistent with recent report on the inhibition of ATP synthesis as a novel therapy for HCC (Geschwind et al. (2002) Cancer Res. 62, 3909 –3913). Taken together, our results suggest that HCC is closely related to liver-specific metabolic process in which mitochondiral events become more evident in hepatocellular carcinogenesis. Molecular & Cellular Proteomics 1.9 677 HUPO First World Congress, November 21–24, Versailles, France 13.31 13.32 Complementary Proteome Analysis of a Human Cancer Cell Line by MALDI-MS and MALDI-MS/MS Human 20S Proteasome: Reference Map, Post-translational Modifications, and Comparative Cancer Cell Lines Analyses E. Claude1, A. Wallace1, D. Gostick1, A. Alaiya2, G. Auer2, and J. Langridge1 Sandrine Uttenweiler-Joseph, Odile Burlet-Schiltz, Ste´ phane Claverol, Elisabeth Neuhauser, Bo Xu, Jean Edouard Gairin, and Bernard Monsarrat 1 Waters Corporation, Floats Road, Wythenshawe, Manchester, United Kingdom; and 2Unit of Cancer Proteomics, Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden Colorectal cancer represents an ideal model system to study development and progression of human tumors. This is because the epithelial cells of the colon follow a systematic process of cellular proliferation, differentiation and adenoma-carcinoma transformation. The p53 gene is actively involved during the different stages of colo-rectal carcinogenesis. In this study, we have examined the role of p53 in a human colorectal cell line (HCT 116) a diploid cell line deficient in DNA mismatch repair mechanism. To understand the role of p53 in the development of colon cancer, we have studied the protein expression profiles in HCT 116 cells with two wild type p53 alleles (p53⫹/⫹) and their p53 knockout counterparts (p53⫺/⫺). Representative 2D gel images were derived from the two samples and were analysed to identify proteins that were significantly up or down regulated. Spots were automatically excised from the gels and then processed on an automated protein digestion robot, used to carry out all the necessary de-staining, alkylation, reduction and protein digestion steps. Automated MALDI-MS was used to rapidly identify many proteins from the digest samples. Some of these proteins however remained unidentified, henceforth a MALDI Q-Tof mass spectrometer was used to provide MS/MS information the extra specificity required to achieve unambiguous protein identification by databank searching.Using this approach it was possible to identify many proteins that were potentially important in the process of colo-rectal carcinogenesis and to localise their positions on specific chromosomes. 678 Molecular & Cellular Proteomics 1.9 Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France The proteasome is an ubiquitous multicatalytic complex, present in the cytoplasm and the nucleus of all eukaryotic cells, which represents the main protein degradation machinery in the cell. Evidence accumulates to show that the proteasome is involved in crucial cellular processes, including cell cycle, apoptosis, morphogenesis, stress response, regulation of intracellular proteins content, removal of abnormal proteins, and major histocompatibility complex (MHC) class I antigen processing. The catalytic core of the proteasome is a barrel-shaped complex called 20S proteasome, which is composed of 28 subunits assembled into two outer ␣ rings and two inner  rings containing 7 subunits each. The outer a subunits regulate the entrance of denatured proteins into the catalytic cavity formed by the  subunits. Three  subunits (1, 2, and 5) per ring possess a catalytic active site and are responsible for three major peptidase activities (trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide hydrolyzing). The catalytic activities of 20S proteasome may be altered by the cellular origin and/or environment of the proteasome. It is known for example that, in the presence of the cytokine ␥-interferon, the three catalytic  subunits of mammalian 20S proteasomes are replaced by three other catalytic subunits, leading to modified peptidase activities. Moreover, post-translational modifications of proteasome subunits, such as phosphorylations, have been shown to affect proteasomal catalytic activities. The aim of our work is to understand the effects of cellular environment on 20S proteasome structure/activity relationships and their consequences on MHC class I tumor antigen processing by proteasome. Here we present the study of the subunit composition of human 20S proteasomes of various cellular origins by proteomics. Post-translational modifications have been characterized using mass spectrometric based strategies. The separation of 20S proteasome subunits by 2D gel electrophoresis and their identification by MALDI-TOF mass spectrometry analyses combined with database searches, provided a reference map of human 20S proteasome subunit composition. This study was performed with 20S proteasome purified from human erythrocytes and allowed us to identify all expected a and b subunits. Moreover, these analyses revealed the presence of numerous isoforms associated to most subunits, including the catalytic subunits. Post-translational modifications, including phosphorylation and N-acetylation, were identified by a combination of chromatographic techniques and mass spectrometry (MALDI-TOF and nanospray MS/MS) for the various isoforms of one a subunit. Comparison of subunit composition maps obtained with human 20S proteasomes purified from various cellular origins (normal versus cancer cell lines) showed quantitative differences for the catalytic subunits and for a number of isoforms. HUPO First World Congress, November 21–24, Versailles, France 13.33 13.35 Differential Cell Surface Protein Expression on Normal and Neoplastic Humal Prostate Cells and Their Regulation by Interferons Proteomic-based Approach for the Identification of Early Tumor Markers Associated with Hepatocellular Carcinoma Soren Naaby-Hansen, Claire Hastie, Akunna Akpan, Malcolm Saxton, Rainer Cramer, Kohji Nagano, and Jonn R. Masters Ludwig Institute For Cancer Research, Royal Free and University College Medical School, London, United Kingdom The cell surface protein expression of a normal prostate epithelial cell line and a prostate cancer cell line derived from the patient were investigated by a combination of vectorial labeling with sulfo-biotin derivatives, 2D gel electrophoresis, avidin blotting, affinity chromatography, and mass spectrometry analysis. Several proteins sere solely detected on the surface of either the cancer or the normal cell line, and many others were found to be expressed at different levers between two cell line. The repertoire of proteins available for biotin labeling changed both qualitatively and quantitatively when the cells were grown in the presence of either or both the therapeutic agents INF-␣ and INF-␥. Certain cell surface proteins including gp96 and Annexin II, were differentially regulated in normal and cancer cell lines by these interferons. Surface exposed Annexin II levels sere specifically down-regulated by INF-␥, while the total cellular levels were unaffected by cytokine treatment. INF-␥ mediated down-regulation of surface exposed Annexin II, which captures and creates a reservoir of surface proteases such as plasmin and procathepsin B, significantly reduced the invasive potential of prostate epithelial cells in cell invasion assays. Our data thus provides a potential explanation for the reduction of metastatic potential observed in response to treatment with this cytokine. Kapil Dua1, Franc¸ ois Le Naour1, Jorge A. Marrero2, David E. Misek3, Christian Bre´ chot 4, Samir M. Hanash3, and Laura Beretta1 1 Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, 48109-0666; 2Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, 48109-0666; 3 Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, 48109-0666; and 4INSERM U.370, Necker Hospital, 75015 Paris, France The incidence of hepatocellular carcinoma (HCC), the major histological form of primary liver cancer, has substantially increased over the past two decades. Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for HCC. We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. We recently reported the identification of 8 proteins including calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A and F1-ATP synthase beta subunit, for each of which, autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p ⬍ 0.05) (Le Naour et al. (2002) Mol Cell Proteomics 1, 197–203). The common occurrence of autoantibody formation to HCC-related proteins may also have value in HCC screening. We are currently investigating the utility of identified autoantibodies for the early diagnosis of HCC among high-risk subjects with chronic HCV infection alone or in combination with cirrhosis. To that effect, two-dimensional gel electrophoresis followed by 2-D Western blotting are used for simultaneous identification of proteins eliciting a humoral response in 36 HCV patients with different degrees of liver fibrosis and cirrhosis. Further prospective patient follow-up will allow validation of these markers for the early diagnosis of HCC. 13.34 Identification of Tumor Antigens That Elicit a Humoral Response to Dendritic Cell-based Immunotherapy in Patients with Melanoma Kapil Dua1, Hong Wang2, Franc¸ ois Le Naour1, Jim Mule3, Samir M. Hanash2, and Laura Beretta1 1 Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, 48109-0666; 2Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, 48109-0666; 3Department of Surgery, University of Michigan, Ann Arbor, Michigan, 48109-0666; and 4University of Michigan, Ann Arbor, Michigan, 48109-0666 We have utilized a proteomic approach to determine whether the humoral response to tumor antigens observed in melanoma patients was modified following dendritic-cell based immunotherapy. Two-dimensional gel electrophoresis was used for simultaneous separation of individual proteins from tumor tissue or cell lines. Proteins eliciting a humoral response in melanoma patients were identified by 2-D Western blotting using sera obtained before and after immunotherapy treatment, followed by mass spectrometry analysis. We identified proteins, for each of which, autoantibodies were absent in sera from healthy individuals but detected in melanoma patients, in different amounts before and after immunotherapy. The identification of these tumor antigens may have utility in melanoma diagnosis or in establishing an appropriate dendritic-cell based vaccine. Molecular & Cellular Proteomics 1.9 679 HUPO First World Congress, November 21–24, Versailles, France 13.36 13.37 Proteomic Characterization of the HCV NS5A Natural Mutants Isolated from Interferon Responder Versus Non-responder Patients with HCV Proteome Differential Display of MCF-7 Cell Lines with High Capability for Active Dissociation of Hoechst 33342 and Wild Type Alison Elafros1, Christian Bre´ chot2, Samir M. Hanash3, and Laura Beretta1 Olga V. Tikhonova1 and Vera V. Levina2 1 Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, 48109-0666; 2INSERM U.370, Necker Hospital, 75015 Paris, France; and 3Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, 48109-0666 Hepatitis C virus (HCV) is a positive-stranded RNA virus, which exhibits marked viral heterogeneity. Therapy using interferon alpha has been shown to be effective in chronic hepatitis C infection, but only 30% to 40% of treated patients show a sustained virologic response. The polyprotein precursor is co- and post-translationally processed by both cellular and viral proteases to yield mature, structural and nonstructural proteins. We have shown that the HCV non-structural NS5A protein inhibits the antiviral activity of interferon alpha (Podevin et al. (2001) Hepatology 33, 1503–1511; Girard et al. (2002) Virology 295, 272–283). We isolated full-length NS5A sequences, from patients with or without response to interferon therapy and combined transient and stable expression of these NS5A mutants in a human hepatocytic cell line (Huh7). We analyzed and compared the phosphorylation and stability of the various NS5A mutants. Several NS5A isoforms were identified by twodimensional gel electrophoresis. Interestingly, the NS5A proteins isolated from non responder patients, migrated into two major spots with pI values of 5.57 and 5.43 respectively, whereas the NS5A proteins isolated from responder patients presented two additional spots with pI values of 5.29 and 5.16 respectively. Using pretreatment of Huh7 protein extracts with calf intestinal phosphatase, we demonstrated that the two additional, more acidic, spots correspond to hyper-phosphorylated isoforms of NS5A. This result demonstrates distinct phosphorylation patterns among the NS5A mutants. 1 Institute of Biomedical Chemistry RAMS, Moscow, Russia; and 2St. Petersburg Institute of Nuclear Physics, St. Petersburg, Russia Living cells are capable to remove noncovalently binding molecules from DNA. This process of active dissociation or ’DNA clearing’ from noncovalently bound agents in living mammalian cells was studied on the model of the vital fluorescent bisbenzimidazole dye Hoechst 33342 and cytofluorimetric technique. It was showed that DNA clearing is one of the mechanisms of multidrug resistance in cancer cells. The method of stepwise selection with increasing concentrations of Hoechst 33342 was used to develop a set of Hoechst 33342 resistant MCF7 human breast adenocarcinoma cell lines. The cell lines with enhanced level of DNA clearing were found to be 300 fold more resistant to toxic action of Hoechst 33342 and netropsin and 20 fold more resistant to etoposide. To recognise the proteins involved in DNA clearing we have studied differences between proteomes of parent MCF7 line, mutant cell line MCF7 HoeR-3 in early stages of selection and strong resistant MCF7 HoeR-7 mutant cell line using the conventional 2D-EF technique. A comparison of corresponding 2D gels shows differences at several protein spots, being more expressed in strongly resistant mutant or vice versa. Some of differential proteins were identified by MALDI-MS. 13.38 BD PowerBlotTM Western Array Analysis of Protein Expression in Differentiated SH-SY5Y Neuroblastomas M. T. Wilson1, J. K. Stevenson1, E. B. Heywood1, A. M. Siemers1, A. L. Claxon1, B. J. Nelson1, C. H. Keith2, and R. Campos-Gonzalez1 1 BD Transduction Laboratories, Lexington, Kentucky; 2Cellular Biology, University of Georgia, Athens, Georgia; 3PNNL, Richland, Washington 99352; and 4Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan A variety of in vitro models of neuronal differentiation and survival have been developed for studying the protein expression changes that occur during these processes. Few studies have compared the global changes in protein expression that occur as a result of using different differentiation factors, such as staurosporine vs NGF. In this study, we used BD Transduction Laboratories Western Array Screening Service (BD PowerBlotTM) to identify protein expression changes for more than 800 proteins in SH-SY5Y cells exposed to NGF for 24 hrs and staurosporine for 16 hrs. Our results show that NGF exposure caused a modest increase in the number of neurite-bearing cells, which correlates with alterations in the expression of some proteins. Staurosporine caused more robust differentiation of SY5Y cells, and this correlates with changes in the expression of many protein families. However, staurosporine also caused detachment of a subpopulation of cells from the substratum, and led to staurosporinespecific decreases in the expression of essential enzymes, cytoskeletal components, and calcium signaling proteins. Many of the proteins expressed after staurosporine-induced differentiation are also expressed during development of the rat cerebrum, however some unique protein expression changes found in staurosporine-differentiated cells do not occur in the rat cerebrum. Thus, BD PowerBlotTM screening analysis is a valuable tool for examining protein expression profiles for pathways involved in neuronal differentiation and death. 680 Molecular & Cellular Proteomics 1.9 HUPO First World Congress, November 21–24, Versailles, France 13.39 13.41 Two-dimensional Gel Electrophoresis Map for Human Stomach Tissue Application of Fluorescence Dyes in a Linkage of Laser Microdissection and Two-dimensional Gel Electrophoresis: As a Tool for Cancer Proteomic Study Seung Uook Lee, Na-Young Ha, Jina Kim, Jin Sook Ahn, Yu Na Kim, Bok Im Cho, Sung-Jo Kang, Jae Won Kim, and Chang Won Lee Division of Life Science, Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, South Korea Stomach cancer is one of the most common cancers in Korea both in men and women. Also, stomach is one of the leading causes of cancer death in the world. We recently reported the proteome analysis of human stomach tissue (Electrophoresis (2002) 23, 2513–2524), using two dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). We have successfully identified 243 protein spots (168 spots acidic map and 75 spot in basic map) from the 2-DE gel of human stomach tissue. Here, we update previously published in addition to urea has been found to further improve solubilization. With these result, we could be detected many low-abundance proteins and high molecular proteins. Second, to improve success rate of protein identification in this study, we performed protein identification using reversed-phase nano column. The use of reversed-phase nano column improved the concentration of extracted peptides and the purification of peptide mixture. Using this approach, 2-DE map have been established for soluble protein of human stomach tissue. 174 protein spots (104 spots acidic map and 71 spot in basic map) were identified newly. The 2-DE maps resulting from this study will server important resources in studies on the differential protein expressions of human stomach cancer and also other human stomach pathologies. Tadashi Kondo, Masahiro Seike, Yasuharu Mori, Kazuyasu Fujii, Tesshi Yamada, and Setsuo Hirohashi Cancer Proteomics Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan The combination of laser microdissection and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used for proteome analysis on specific populations of cells in cancer tissues. However, because of low sensitivity of spot detection method, silver staining, the microdissection to obtain enough amount of protein for 2D-PAGE is too laborious and only restricted numbers of protein spots were visualized. As a consequence, this technology looks impractical for direct clinical applications and had a limited impact on cancer study. To solve these problems, we developed new application in which sensitive fluorescent dyes label the proteins extracted from microdissected tissues prior to 2D-PAGE separation. In this application, small amount of proteins, less than 6.6 g, was enough to generate a 2D profile with approximately 1,500 protein spots. For accurate quantification of spot intensity, different samples were labeled with different fluorescence dyes and run in a single 2D gel. This technique was applied to compare the proteome of normal intestinal epithelium and adenoma tissues of Min mice. Among 1,500 spots, 37 spots changed their intensities reproducibly: 27 spots showed increased intensity and 10 spots decreased in adenoma tissues compared with their normal counterparts. Mass spectrometric analysis and subsequent database search successfully identified 4 of them, which included prohibitin, 14 –3-3, tropomyosin 3 and Hsp84. These results indicate that the fluorescence labeling of proteins from microdissected tissues prior to 2DPAGE is a powerful tool in cancer proteomic study. 13.40 Proteome Analysis of Gastric Cancer: Overexpression of Nucleoside Diphosphate Kinase A in Human Gastric Cancer Tissue Sung-Jo Kang, Na-Young Ha, Jina Kim, Jin Sook Ahn, Yu Na Kim, Bok Im Cho, Seung Uook Lee, Jae Won Kim, and Chang Won Lee Division of Life Science, Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701, South Korea Gastric cancer is the most common cancer in Korea and Asian countries. Although incidence rates in western world are much lower than Asia, gastric carcinoma is still a significant worldwide health burden, second only to lung tumors as a leading cause of cancer deaths. In this study, in order to develop novel markers for gastric cancer, 70 cases of gastric cancer tissue were analyzed by two-dimensional electrophoresis, and stained with silver nitrate. The images of stained gels were analyzed with aid of software, PDQuest. Of the 70 samples, 16 samples (22.86%) showed higher expressions of the spot 6117 in cancer tissue. The spot was excised from preparative gels, digested by trypsin, and subjected to peptide mass fingerprinting. The spot was identified to be nucleoside diphosphate kinase A, with 10 matching peptides which corresponds to a sequence coverage of 53%. Nucleoside diphosphate kinase A is a ubiquitous enzyme responsible for the synthesis of most cellular non ATP nucleoside triphosphates. Beside their role in nucleotide synthesis, these enzymes present additional functions, possibly independent of catalysis, in processes such as differentiation, cell growth, tumor progression, metastasis and development. Molecular & Cellular Proteomics 1.9 681 HUPO First World Congress, November 21–24, Versailles, France 13.42 13.43 Proteomics of Renal Disorders: Urinary Proteome Analysis by Two-dimensional Electrophoresis and MALDI-TOF Mass Spectrometry Isolation of Rheumatoid Arthritis-specific Proteins Using Proteomic Analysis Yadunanda Kumar1, Nageshwar Rao Venkata Uppuluri1, Kishore Babu2, Kishore Phadke3, Prasanna Kumar2, Sudarshan Ballal4, and Utpal Tatu1 1 Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India; 2Department of Endocrinology, M.S. Ramaiah Medical College Hospital, Bangalore 560 054, India; 3 Department of Pediatrics, St. John’s Medical College Hospital, Bangalore 560 034, India; and 4Manipal Institute of Nephrology and Urology, Manipal Hospital, Bangalore 560 017, India The proteomes of urinary samples from patients with different renal conditions were analyzed by 2-DE and MALDI-TOF technology. Samples from three different renal conditions namely kidney failure, nephrotic syndrome and microalbuminuria, were included in the analysis. Apart from the presence of albumin, the profiles of protein spots found in these urine samples were quite distinct. While kidney failure patients showed predominantly low molecular weight proteins, the nephrotic syndrome patients showed an abundance of relatively high molecular weight proteins clustering in the acidic range of the 2-D gels. Two different protein spots from kidney failure patients, four from nephritic syndrome and three from microalbuminuria patients were identified by in-gel protease digestions and analysis of resulting peptides by MALDI-TOF. The proteins identified were albumin, ␣-1-antitrypsin, ␣-1-acid glycoprotein 2, Zn-␣-2-glycoprotein and ␣-1microglobulin. Among these only one was common between the proteomes of renal failure and nephrotic syndrome patients. Among the limited proteins found in microalbuminuria patients, three were common with the proteome of nephrotic syndrome. Overall profiles were, however, quite different. In all, our study showed that urinary proteomes of different renal conditions were different and emphasized the potential of urinary proteome analysis to augment existing tools in the diagnosis of renal disorders. Deok Ryong Kim1, Young-Sool Hah1, Eun-Hye Cho1, Yun Jong Lee2, and Choong Won Kim1 1 College of Medicine, Gyeongsang National University, 92 Chilam-dong, JinJu, KyeongNam, South Korea 660-751; and 2 Department of Biochemistry, Department of internal medicine, Gyeongsang National University, 92 Chilam-dong, JinJu, KyeongNam, South Korea 660-751 Rheumatoid Arthritis (RA), an autoimmune disease with chronic inflammation and destruction of cartilage and bone, has a prevalence of 1% over the world. The immunological and molecular mechanism in RA pathogenesis has not been elucidated clearly, although many efforts were involved in the research of RA. Cytokines (IL-10, TNF␣, etc.) that regulate immune cell proliferation have been thought the key controller in RA pathogenesis. In fact, blocking the function of TNF␣ using antagonist antibodies prohibits the progression of RA. However, the role of such cytokines in RA pathogenesis is not clearly demonstrated. The immune response to the self antigens in RA is triggered by the activation of potential self-reacting B or T cells with mimicry antigens that might be derived from viral or bacterial infection. Here we are trying to isolate RA-specific proteins from comparative proteomic analyses of proteins obtained from Korean RA, Osteoarthritis, or other arthritis patients. We separated total cell proteins (about 2,000) or subcellular (soluble or insoluble) proteins extracted from patients in two-dimensional gel electrophoresis and analyzed the pattern of protein expression using PDQuest software. We found that some proteins were uniquely expressed in RA patients and expression of many proteins either increased or decreased. Isolation of RA-specific proteins and new autoantigens will provide some insights into understanding the molecular mechanism in RA pathogenesis and separating target proteins for new drug development. 13.44 Discovery, Purification, and Identification of Biomarkers from Complex Samples by Combining ProteinChip姞 Array Analysis with Micropurification Strategies Rosa I. Viner, Siyu Fu, Ning Tang, and Scot R. Weinberger Ciphergen Biosystems, Inc., Fremont, California 94555 In this study we present a novel approach to biomarker identification via Retentate Chromatography™-Mass Spectrometry (RC-MS) on the SELDI ProteinChip platform. The work combines RC-MS protein profiling and spin column micropurification followed by gel electrophoresis using limiting amounts of starting material. This strategy consists of four main components: biomarker discovery by SELDI; biomarker enrichment by combining micropurification with electrophoresis; recovering purified proteins using passive gel elution; and confirmation of protein identity utilizing on-spot proteolysis followed by tandem ms analysis. We have used Q-HyperD姞F anion exchange micro-spin columns for pre-fractionation and RC-MS to monitor human Parathyroid Hormone (PTH) spiked into Cerebral Spinal Fluid (CSF). RC-MS analysis has shown the presence of this protein in an organic fraction which strongly bound to reversed phase arrays, even after 50% acetonitrile wash. The protein was then purified by 4 –20% Tris-Glycine SDS PAGE and detected by a reverse negative staining procedure. The PTH band was excised from the gel, destained and the protein was eluted as described in (1) with minor modifications. The purified protein has shown the same molecular weight and chromatographic properties on the H50 array as PTH and its identity was confirmed with high confidence after AspN on-spot digestion through a combination of peptide mass fingerprinting by SELDI-TOF MS and peptide sequencing by SELDI-QqTOF MS/MS. We have successfully applied this technique to identify potential biomarkers from other complex biological systems. 1. Cohen, S. L., and Chait, B. T. (1997) Anal. Biochem. 247, 257–267 682 Molecular & Cellular Proteomics 1.9 HUPO First World Congress, November 21–24, Versailles, France 13.45 14.1 Post-translational Modification of Serum Proteins Underlies the Mass Spectral Fingerprinting of Disease Proteomic Analysis of Acholeplasma laidlawii Mutants Resistant to Tetracycline and Fluoroquinolone John Marshall, Peter Kupchak, Weimin Zhu, Jason Yantha, Tammy Vrees, Shirley Furesz, Kelly Jacks, Chris Smith, Inga Kireeva, Rulin Zhang, Miyoko Takahashi, Eric Stanton, and George Jackowski Sergei Moshkovskii, Vadim Govorun, and Eugene Goufman Synx Pharma, 1 Marmac Drive, Toronto, Ontario, M9W 1E7 Canada The mass distribution and intensity of poly-peptides from human sera obtained using mass spectrometry has served as a fingerprint used to diagnose disease. However, the mechanism underlying mass spectral diagnosis has not been demonstrated. We extended the method to obtain a serum fingerprint of myocardial infarction patients which forms the basis of a powerful diagnostic tool that rapidly and conclusively confirms the presence of myocardial infarction. We show that in general mass spectral diagnosis works on the principle of detecting post-translational modifications of major serum proteins effected by disease associated activities in the blood. In the case of myocardial infarction, the MALDI-TOF spectra of peptides collected by C18 reversed-phase chromatography form a diagnostic pattern resulting from the post-translational modification of complement C3 ␣ chain to release the C3f fragment and cleavage of fibrinogen to release the alpha peptide. We used time course and PMSF studies to demonstrate that the peptides that form diagnostic patterns in the serum result from polypeptides that were continually being generated by serinecentered endo-proteinases. However, we also show that the peptides cleaved from serum proteins by endo-proteinases are themselves in turn degraded by N-terminal exopeptidase(s), i.e., aminopeptidase. Thus the mass spectral patterns that form the basis of diagnosis reflect a balance of the proteinase and aminopeptidase specificity and activity in the sera. On this basis we conclude that MALDI-TOF, or other mass spectral fingerprinting, of sera may reflect the interaction of disease-associated molecules with the proteins of the blood. 13.46 Analysis of Complex Autoantiboy Repertoires by SELDI-TOF V.N. Orekhovich Institute of Biomedical Chemistry, Moscow, Russia Acholeplasma laidlawii was firstly investigated as a good experimental model for membrane transport studies of antimicrobial drugs like fluoroquinolones, tetracycline and similar compounds. However, the role of mycoplasma plasma membrane transport systems in the resistance phenomena has not yet been studied. The 2D electrophoretic (2DE) maps of Acholeplasma laidlawii cell and membrane proteins were obtained for the comparative analysis of 2DE maps of mutants resistant to tetracycline and fluoroquinolones and wild type. For the solubilization and separation of Acholeplasma laidlawii plasma membrane proteins in the first dimension (IPG-strips pH 3–10, Bio-Rad) we used detergents, such as CHAPS (ICN), Triton X-100 (Bio-Rad), Noctylglucoside (ICN), NP-40 (ICN) and Octylpol (Alexis, USA). For the second dimension 5–18% gradient SDS-PAGEs were used. Silver staining procedure was performed for the visualization of separated proteins. Analysis of the 2DE gels was carried out using Melanie II software. Several 2DE of Acholeplasma laidlawii were obtained using different detergents. NP-40 was the most effective detergent for the solubilization and separation of Acholeplasma laidlawii cell proteins. In contrast, Octylpol has been chosen as optimal detergent to solubilize membrane proteins. There were 670 individual membrane protein spots on gel after silver staining. Analysis of Acholeplasma laidlawii mutants resistant to tetracycline and fluoroquinolones 2D-PAGE maps revealed increasing expression of several proteins which were suggested to be involved in the resistance mechanism. Both mutants resistant to tetracycline and fluoroquinolones had 4 protein spots of molecular weight about 9 –14 kDa with increased intensity. Using MALDI-MS of tripsin digests of these spots it has been found that three of them occur derived from the same gene and one protein was different. Selected fragments of these two individual proteins were investigated using ESI-MS-MS in order to determine amino acid sequence. Basing this information PCR primers were constructed and corresponding genes were cloned and sequenced using conventional technique known in the art. F. H. Grus, S. C. Joachim, and N. Pfeiffer Department of Ophthalmology, University of Mainz, Mainz, Germany Normal sera contain a large number of naturally occurring auto-antibodies which can mask important disease-associated ones. In previous studies we could show the diagnostic potential of the analysis of auto-antibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, conventional Western blotting procedures remain very time-consuming limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDITOF (surface-enhanced laser desorption/ionisation in time-of-flight mass spectrometry) technology (Ciphergen, Fremont, California) for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The micro-scale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biological activated surfaces that permit antibody capture studies. In the present study, we could show complex on-chip antibody-antigen reactions. At higher molecular weights (⬎30 kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular weights, the Western blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of auto-antibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex auto-antibody distributions for diagnostic purposes and early detection of autoimmune diseases could be made possible. Molecular & Cellular Proteomics 1.9 683
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