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计划产生了大量信息，催生了新技术，并催生了一种新型生物学，它试图从对生物系统和从基因到蛋白质的途径的全球分析中建立生物学知识。迫切需要开发一种全球评估手段，最终在功能蛋白质水平上对生物学有更清晰的理解。由于蛋白质的活性和周转都与蛋白质的翻译后修饰(如泛素化、磷酸化等)密切相关，因此必须开发新的技术来进行大规模的蛋白质翻译后修饰的鉴定、表征和量化。 泛素化参与了许多细胞过程，包括依赖于ATP的细胞蛋白质的选择性降解、染色质结构的维持、基因表达的调节、受体介导的内吞作用、应激反应和核糖体的生物发生。我们建议开发一系列新的技术，用于从细胞和组织中大规模(最终是全球)的生物样本中分离、鉴定和定量泛素结合物。这一新策略将是一种高度集成的方法，利用亲和标签(6xHis)进行泛素结合物的全球分离，利用多维色谱-串联质谱仪(LC/LC-MS/MS)进行肽/蛋白质序列分析，以及使用同位素编码亲和标签(ICAT)方法进行准确的蛋白质定量。此外，我们将在酿酒酵母中确定泛素中每个赖氨酸残基在多泛素链形成中的作用。这将通过创建7个胰酶泛素标志性多肽来实现，这些多肽标记有跨越每个赖氨酸残基(K6、K11、K27、K29、K33、K48和K63)的稳定同位素。这将允许通过刚才提到的任何赖氨酸残基来定量形成多链。

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.