Understanding how genomes control the physiology of biological systems requires detailed analysis of the proteins produced. In the past several years, technical refinements to mass spectroscopy and bioinformatics methods have increased the power of analyzing protein populations (proteomics) by another order of magnitude, making it vital to research projects at the Medical College of Ohio and neighboring institutions. The purpose of this proposal is to obtain a liquid chromatography - tandem mass spectroscopy (LC/MS/MS) instrument, along with the core accessories required to allow its most efficient use by NIH-funded major users, and a variety of minor users from the Medical College of Ohio, University of Toledo, and Bowling Green State University. Unlike DNA sequencing, efficient proteomic analysis by LC/MS/MS requires close interactions between the research team and core laboratory personnel. This is particularly true of LC/MS/MS, in which a population of tryptic peptides are resolved, released from a gated ion trap, fragmented by collision with atoms of an inert gas, and sequenced by analyzing of the mass fingerprint of the resulting ions. Laboratory space to house the proposed equipment, and expertise required to ensure its optimal use, are already in place at the Medical College of Ohio. Major and minor users of the proposed LC/MS/MS equipment have been identified. The NIH-funded projects of the eight major users include characterization of the fungal pathogen and potential bioterrorism agent Coccidioides immitis, the link between reactive oxygen species-dependent inactivation of Na + ATPase and pathological changes in cardiac myocytes, characterization of proteins interacting with Na + ATPase, the role of the CEACAM1 cell adhesion molecule in insulin metabolism, regulation of replication of the potential gene therapy vector adeno-associated virus (AAV), characterization of the MAP kinase inhibitory protein RKIP, investigating the role of Rab GTPases in controlling cell growth, and characterizing the endocytosis and recycling of the GPI-anchored folate receptor. All of these projects, and others as well, will be substantially strengthened by the requested equipment.
