Core B: Proteomics and Protein Analysis Core

项目总结

PROJECT SUMMARY Core B will provide proteomics and protein analysis services for PPG projects. Throughout the last funding cycle, the protein analysis component of Core B has significantly expanded to meet the needs of the individual projects in the PPG. In addition to mass spectrometry (MS)-based footprinting technology for the mapping of protein-protein and protein-nucleic acid interactions proposed in the previous application, we have added the following new services to the Core: 1) the AKTA-fast protein liquid chromatography (FPLC), which has played a critical role in obtaining purified recombinant proteins for biochemical/biophysical characterization for former Project 3 and antibody production for former Project 2; and 2) the EnSpire multimode plate reader to provide a new service for highly sensitive, rapid and robust monitoring of protein-protein and protein-nucleic acid interactions. MS-based proteomic approaches enabled us to identify new protein-protein interactions and posttranslational modifications, and have thus been instrumental for advancing the research of former Projects 1 and 2, respectively. The above technologies will continue to play an important role in accomplishing the research objectives of the PPG projects proposed in this competitive renewal. In particular, Specific Aims of the Proteomics and Protein Analysis Core are: Aim 1: to perform MS-based protein footprinting experiments to identify contact amino acids in the context of large biologically relevant nucleoprotein and protein-protein complexes; Aim 2: to develop novel applications of the EnSpire multimode plate reader for biochemical characterization of protein-nucleic acid and protein-protein interactions using homogeneous time-resolved fluorescence energy transfer (HTRF), AlphaLISA, and AlphaScreen (ALPHA) based technologies; Aim 3: to provide a column chromatography service using AKTA-FPLC to assist individual PPG projects to isolate low abundant native protein-protein complexes from mammalian cells and purify mg quantities of poorly soluble recombinant proteins from E. coli for their subsequent biochemical and biophysical characterization; Aim 4: to conduct MS-based proteomics experiments to identify interacting partners and protein posttranslational modifications. Taken together, Core B will provide the wide range of protein analysis services. Importantly, these services will be utilized by all four PPG projects.