PROTEOMICS

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The Protein Chemistry and Mass Spectrometry ('Proteomics') Core will be under the administration of the Buck Institute's Chemistry Program whose director is Dr. Gibson. Dr. Gibson has over 20 years experience in mass spectrometry and the analysis of proteins and other macromolecules. In the last seven years, this group has carried out extensive studies in mitochondrial proteomics as well as developing new methods to study posttranslational modifications such as 3-nitrotyrosine, phosphorylation and cysteine oxidation. The proteomics facilities in the Core include extensive mass spectrometry, chromatography, and protein chemistry components that are well suited to provide the expertise and methodology needed to carry out the protein chemistry experiments described in Projects 1-3. The resources and knowledge of the Core staff include methods for mitochondrial proteomics, protein identification, chemistry and quantitation, Moreover, methods have been described for the rapid isolation and analysis of large complexes (e.g., mitochondrial electron transport chain, or ETC), and the analysis of protein posttranslational modifications (PTMs), including oxidative damage, protein phosphorylation, and changes in subunit stoichiometries. Therefore, our aims for this Core are to work closely with the PL's of the individual Projects to provide detailed analysis of proteins, i.e., mitochondrial OXPHOS complexes I-V, p53 and tau as well as other proteins, and changes they undergo in response to the manipulation of genes/enzymes that affect the overall redox status, or 'oxidative stress' of the various age-related disease cell and/or animal model systems. The specific aims of the Proteomics Core in relation to the three Projects in this Program Project are: Andersen, Project 1: We will examine in detail both oxidative damage (both reversible and irreversible) and changes in phosphorylation among the 46 subunits of mitochondrial Complex I (and possibly other ETC complexes and mitochondrial proteins) that accompany acute and/or chronic depletion of glutathione pools in an transgenic anti-GCLmouse and a rat dopaminergic N27 cell line. Campisi and Benz, Project 2: We will examine changes in p53 phosphorylation and cysteinyl redox status (i.e., glutathionylation, cysteic acid, etc.) in human and mouse epithelial fibroblasts (MEFs) when SOD2, glutathione and p53 expression levels have been manipulated and correlated to changes in either p53 levels or DNA binding. Melov Project 3: We will examine changes in the phosphorylation state of tau in a constituitive and, at later stages, an inducible sod2 nullizygous mouse model as a consequence of age and oxidative stress.