A Proteomics Approach To Protein Ubiquitination

描述（由申请人提供）： 包括人类基因组计划在内的全球基因组倡议产生了大量信息，催生了新技术，并促进了一种新型生物学的出现，这种生物学试图通过对生物系统和从基因到蛋白质的途径的全球分析来建立生物学知识。迫切需要开发全球评估手段，最终在功能蛋白质一级提供对生物学更清楚的了解。因为蛋白质活性和周转都与蛋白质翻译后修饰密切相关（例如，泛素化、磷酸化等），必须开发新的技术以允许大规模鉴定、表征和定量蛋白质翻译后修饰。 泛素化参与许多细胞过程，包括细胞蛋白质的ATP依赖性选择性降解、染色质结构的维持、基因表达的调节、受体介导的内吞作用、应激反应和核糖体生物合成。我们建议开发一系列新的技术，用于在大规模（最终全球）的生物样品中从细胞和组织中分离、鉴定和定量泛素缀合物。这种新的策略将是一种高度集成的方法，利用亲和标记（6xHis）的泛素缀合物的全球分离，多维色谱-串联质谱（LC/LC-MS/MS）的肽/蛋白质序列分析，和同位素编码的亲和标签（ICAT）的方法，准确的蛋白质定量。此外，我们将确定在酿酒酵母中的作用，每个赖氨酸残基在泛素相对于聚泛素链的形成。这将通过产生七个胰蛋白酶泛素标记肽来实现，这些肽用跨越每个赖氨酸残基的稳定同位素（K 6、K 11、K 27、K 29、K 33、K 48和K63）标记。这将允许通过刚才提到的任何赖氨酸残基来定量多链形成。

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.