5.6 Abstract - PROTEOMICS (Core Group B) The Proteomics Shared Resource (Core Group B) offers a state-of-the-art infrastructure for protein analysis from simple protein ID to global analysis, which is tailored to the specific needs of Cancer Center investigators. The Core technologies include methods for upfront sample fractionation and back-end protein identification by mass spectrometry (MS) and database searching. Core MS instruments include Thermo LTQ, LTQ Orbitrap XL and LTQ Orbitrap Velos Pro systems, and Bruker HCTUltra and MALDI Autoflex II TOF/TOF systems. Routine services include protein/peptide molecular weight determination by MALDI-MS, identification of gel separated proteins and mapping of post-translational modifications (phosphorylation, ubiquitylation, acetylation, glycosylation) by tandem MS, and global quantitative proteomic and phospho-proteomic profiling by 2D or 3D LC-MS/MS methodology. Routinely employed methods of quantification include stable isotope- based methods (SILAC, 15N-SIL, AQUA, iTRAQ, TMT) and various label-free techniques (normalized spectral abundance factor, peak intensity measurements). The Core utilizes a number of advanced data analysis tools, including the Sorcerer Enterprise Server and the IP2/Census Server. Cancer Center investigators are utilizing these technologies in a variety of ways, such as identifying novel components of protein complexes and protein-protein interactions, mapping functionally important post-translational modifications such as phosphorylation and ubiquitylation, and comprehensively profiling quantitative differences in protein abundance in cancer versus normal cells. To facilitate these projects, Core personnel routinely meet and interact with investigators to provide guidance with project design, sample preparation, and data analysis. In addition, the Core engages in outreach activities geared toward training investigators in proteomic technology. In part due to outreach activities, the Core is broadly used, with services provided to 27 Cancer Center members in the past year. The Core's future activities will focus on maintaining high quality services as well as the exploration, testing, and implementation of novel technologies that will benefit the Center's research programs. Efforts in planning toward these goals include the implementation of a targeted proteomics workflow using selected ion monitoring (SRM) on a triple quadrupole mass spectrometer. In addition, the Core will work toward the implementation of improved methods for profiling post-translational modifications (e.g. glycoproteomics and global profiling of ubiquitylated proteins by affinity enrichment). Data analysis capabilities will also be extended, implementing MaxQuant integrated with Bioconductor, and finding a computational solution for automated subtraction of unspecific background proteins identified in protein interaction screens by affinity purification and MS. In the past funding period, 45 Cancer Center members used the Proteomics Shared Resource and the Core contributed critical data to at least 68 cancer-relevant publications and 38 grant applications.
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