Research Project
10249125
PROJECT SUMMARY The Proteomics and Metabolomics Facility, supported by the Cancer Center Support Grant (CCSG) awarded to The Wistar Institute, provides a comprehensive set of proteomics and targeted quantitative metabolite assays to the Wistar Cancer Center membership as a primary goal. Resources of this Facility are also available to investigators in other Cancer Centers and other academic investigators as a secondary goal. Dr. Tang?s role as the Facility Managing Director is to support cancer research and other biomedical research by providing expert consultation and state-of-the-art technologies that operate at maximum performance at affordable costs to users. The Managing Director will assist in experimental design, manage Facility staff, perform MS data analyses as needed, and assist in the biological interpretation of results. The Managing Director will also devote substantial effort to optimizing and implementing new methods, update analytical and data analyses methods, and update instrumentation to ensure each project is performed using state-of-the-art methodologies. This is critical because instrumentation, software, and analytical strategies continue to evolve rapidly, and most current and anticipated future projects involve very challenging proteomics and metabolomics problems. Proteomics projects will include: 1) LC-MS/MS analysis of isolated protein complexes with and without chemical crosslinking, 2) in-depth, global, quantitative comparisons of exosomes, secretomes, and cell lysates, and 3) targeted and global quantitative comparisons of posttranslational modifications. Quantitative data will be obtained either using label-free quantitation of integrated MS ion currents, or quantitation using stable isotopes such as SILAC or isobaric tag (TMT or iTRAQ) labeling. The proteome analyses will use optimized methods and state-of-the-art instruments, such as the Thermo Scientific Q Exactive HF mass spectrometer, that can detect and robustly quantify most of the proteins present in complex samples, including whole cell lysates. Metabolomics projects will include quantifying the steady state levels of key metabolites using targeted multiplex MRM-MS on the Sciex QTRAP 5500 hybrid triple quadrupole linear ion trap mass spectrometer. Plans for future development include: 1) expanding depth of proteome analyses to efficiently analyze greater than 90% of gene products expressed by cells and tissues, 2) implementing routine use of chemical cross-linking to identify protein-protein interactions and obtain structural information, 3) implementation of metabolite flux analyses using 13C stable isotope tracer, and 4) implementation of lipidomics analyses. All of these new methods will use the Thermo Scientific Q Exactive mass spectrometers or future next generation instruments. These proteomics and metabolomics analyses will contribute to critical data required to identify protein and metabolite targets, as well as generate hypotheses that are vital for the success of the cancer-related projects described in this application.
