New Technologies for Analysis of Protein Phosphorylation

描述(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。因此，越来越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(如磷酸化和泛素化)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。 可逆的蛋白质磷酸化是一个影响无数细胞过程的普遍事件。除了在正常生理中起关键作用外，蛋白质磷酸化障碍还与许多疾病的病因有关，如糖尿病、癌症和阿尔茨海默病。虽然蛋白质磷酸化是一个热门的研究领域，但大规模分离和鉴定磷酸化位点是一个尚未解决的问题。我们建议开发一系列新的技术，用于从细胞和组织的生物样本中分离、鉴定和定量大规模(最终是全球)通过磷酸化修饰的蛋白质。这一新策略将是一种高度集成的方法，使用强阳离子交换(SCX)层析浓缩磷酸肽，使用纳米级微毛细管液相色谱-串联质谱仪(LC-MS/MS)进行磷酸肽测序，并使用稳定同位素掺入法进行定量磷化蛋白质组学。此外，我们将应用该策略从初级组织和细胞系中识别数万个磷酸化位点。

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. Consequently, there is a growing 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 post-translational modification (e.g., phosphorylation and ubiquitination), new technologies must be developed to allow for the large-scale identification, characterization, and quantification of protein post-translational modifications. Reversible protein phosphorylation is a general event affecting countless cellular processes. In addition to a critical role in normal physiology, malfunctions in protein phosphorylation have been implicated in the etiology of many diseases such as diabetes, cancer, and Alzheimer's disease. Although protein phosphorylation is an intense research area, the large-scale isolation and identification of phosphorylation sites is an unsolved problem. We propose to develop a series of novel technologies for the isolation, identification, and quantification of proteins modified by phosphorylation on a large (ultimately global) scale in biological samples from cells and tissues. This new strategy will be a highly integrated approach using strong cation exchange (SCX) chromatography for phosphopeptide enrichment, nano-scale microcapillary liquid chromatography tandem mass spectrometry (LC-MS/MS) for phosphopeptide sequencing, and stable isotope incorporation for quantitative phosphoproteomics. In addition, we will apply the strategy to identify tens of thousands of phosphorylation sites from both primary tissues and cell lines.