The Proteomics Core (Core B) provides state-of-the-art instrumentation and expertise in analytical proteomics to investigators in this PPG. A unique feature of the Proteomics Core is the availability of all major proteomics analysis technologies, which offers exceptional flexibility in analytical approaches. The Proteomics Core provides 1) identification of proteins in both simple and complex multiprotein samples, ncluding protein components of multiprotein complexes, 2) quantitative analyses of differential protein expression and modification, and 3) analyses of protein modifications. Proteomics Core staff provide consultation on experimental design and sample preparation, as well as digestion, protein and peptide separations, MS analyses and primary data workup. Both electrospray onization (ESI) mass spectrometers for LC-MS-MS experiments and MALDI TOF-TOF instruments are available for MS analyses. DIGE/MS technology includes fluorescent dyes and multiplexed 2D gels with nternal standards for direct quantitative comparisons. Automated workstations are used for protein excision and sample processing, followed by MALDI-TOF MS and TOF/TOF MS/MS and automated database searching with Mascot software for protein identification. The Core also offers relative quantitation with Nterminal reactive differential labeling reagents (iTRAQ and isotopically-labeled phenylisocyanate) for relative quantitation of modified protein forms. For protein identification from multiprotein complexes, multidimensional LC is coupled directly with automated LC-MS-MS, followed by automated database searching with Sequest and Mascot software. Sequest searches are done on a high speed, multiprocessor Linux cluster in the Advanced Computing Center for Research and Education (ACCRE). Sequest-based identifications are evaluated and filtered using open-source tools (Peptide/Protein Prophet) and integrated with a custom-designed database system (CHIPS) for organizing, filtering and comparing data sets. Additional data analysis by SALSA and P-Mod enables identification of posttranslational modifications, adducts or sequence variants from MS-MS data. Project 1 (Peek) will utilize the DIGE/MS differential-display technology, and Project 3 (Cover) will utilize the protein identification technologies from both simple and complex multiprotein samples. Project 2 (Polk) will heavily utilize both services. Analysis of protein modifications is no longer proposed in the revised Projects, but may be used for subsequent/complementary studies.

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National Cancer Institute (NCI)
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Vanderbilt University Medical Center
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