The Proteomics Core (Core B) provides state-of-the-art instrumentation, methodology, and expertise in proteomics to investigators in this PPG. A unique feature of the Proteomics Core is the availability of cutting? edge quantitative proteomics analysis technologies that allows high sensitivity measurements of proteome changes in models of gastric cancer. The Proteomics Core provides 1) identification of proteins in complex samples, 2) quantitative analyses of differential protein expression from in vitro and in vivo samples, and 3) global analyses of protein modifications. Proteomics Core staff provides consultation on experimentaldesign as well as hands on sample preparation, mass spectrometry analysis, and primary data analysis. Multiple high performance mass spectrometers for LC-MS/MS experiments and MALDI TOF-TOF instruments are available for proteomic analyses. For protein identification from complex samples, analysis involves multidimensional LC-MS/MS followed by automated database searching with Sequest software running on a multiprocessor Linux cluster in the Advanced Computing Center for Research and Education (ACCRE). Protein identifications are evaluated, filtered, and compared using custom lDPicker or commercial Scaffold software. Additional data analysis by TagRecon enables identification of unanticipated modifications or sequence variants from MS/MS data. The Core also provides relative quantitation using stable isotope differential labeling strategies, iTRAQ and SILAC, for samples derived from animal models or from cell culture, respectively. Validation of protein expression changes is accomplished using multiple reaction monitoring LC-MS/MS methods. Project 1 (Peek) will utilize the iTRAQ method to measure proteomic changes in gastric epithelial cells from gerbils infected with carcinogenic H. pylori or mutants fed iron-replete and iron-deplete diets. Project 2 (Wilson) will utilize the same approach to determine changes in gastric epithelial cell proteomes upon EGFR activation. In addition, this Project will utilize the SILAC method to measure changes in the phosphoproteome upon EGFR activation in conditionally immortalized cells. Project 3 (Cover) will employ the iTRAQ method to measure differences in H. pylori output strains from gerbils fed high salt or regular diets. Targeted MRM validation will be employed in all studies to confirm specific proteomic changes
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