A Proteomics Approach To Protein Ubiquitination

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DESCRIPTION (provided by applicant): Global genome initiatives including the Human Genome Project have generated enormous amounts of information, spawned new technologies and catalyzed the emergence of a new type of biology which attempts to build biological knowledge from the global analysis of biological systems and pathways from genes to proteins. There is an urgent need to develop means of global assessment that will ultimately provide a more clear understanding of biology at the functional protein level. Because both protein activity and turnover are tied closely to protein posttranslational modification (e.g., ubiquitination, phosphorylation, etc.), new technologies must be developed to allow for the large-scale identification, characterization, and quantification of protein post-translational modifications. Ubiquitination is involved in many cellular processes including the ATP-dependent selective degradation of cellular proteins, the maintenance of chromatin structure, the regulation of gene expression, receptor mediated endocytosis, the stress response, and ribosome biogenesis. We propose to develop a series of novel technologies for the isolation, identification, and quantification of ubiquitin conjugates on a large (ultimately global) scale in biological samples from cells and tissues. This new strategy will be a highly integrated approach utilizing affinity-tagging (6xHis) for the global isolation of ubiquitin conjugates, multidimensional chromatography-tandem mass spectrometry (LC/LC-MS/MS) for peptide/protein sequence analysis, and the isotope-coded affinity tags (ICAT) method for accurate protein quantification. In addition, we will determine in Saccharomyces cerevisiae the role of each lysine residue in ubiquitin with respect to polyubiquitin chain formation. This will be accomplished by creating seven tryptic ubiquitin signature peptides labeled with stable isotopes that span each lysine residue (K 6, K 11, K 27, K 29, K 33, K 48, and K63). This will allow for the quantification of polychain formation through any of the lysine residues just mentioned.