New Technologies for Quantitative Phosphorylation Analysis

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，并促进了生物学一门新学科的出现。系统生物学试图通过对从基因到蛋白质的生物系统和途径的全球分析来建立生物学知识。为了推动这一不断发展的学科，有必要开发一种全球评估方法，最终澄清我们对蛋白质功能的理解。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，因此需要新的技术来大规模识别、表征和量化蛋白质翻译后修饰。可逆的蛋白质磷酸化是影响大多数细胞调控系统的一般过程。磷酸化是维持正常生理的关键；蛋白质磷酸化异常与糖尿病、癌症和阿尔茨海默病等多种疾病的病因有关。蛋白质磷酸化是一个对改善人类健康大有可为的研究领域。根据这笔赠款的前一个周期开发的技术允许从单个样本中快速、灵敏和准确地识别数千个磷酸化位点。这一里程碑式的成就揭示了其他重要的悬而未决的问题。这项提案涉及两个最关键的问题--定量磷酸化图谱和磷酸化位点占有率。对于目标1，我们将制定定量策略，在近全球范围内研究动态磷酸化。这包括将强大的稳定同位素标记战略、新的浓缩方法和新的软件工具与我们现有的平台相结合和整合。对于目标2，我们将进行一系列高度集中的实验，以全面分析酵母细胞和人类细胞系DNA损伤后的磷酸化差异。这些实验将为新开发的技术和软件提供一个具有重要生物学意义的试验场。最后，对于目标3，我们将开发并应用一种策略来确定串联亲和纯化(TAP)酵母文库中所有激酶(87)的基本位置占有率水平。此外，还将检查一组人类激酶的位置占有率水平，以确定DNA损伤和细胞周期依赖性。公共健康相关蛋白的磷酸化是一个对改善人类健康大有可为的研究领域。除了在正常生理中的关键作用外，蛋白质磷酸化功能障碍也被认为是糖尿病、癌症和阿尔茨海默病等多种疾病的致病因素之一。这项提议将提供新的技术，在全球范围内描述不同细胞状态之间的磷酸化事件。

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 discipline in biology. Systems biology attempts to build biological knowledge from the global analysis of biological systems and pathways from genes to proteins. To fuel this growing discipline, there is a need to develop means of global assessment that will ultimately clarify our understanding of protein function. Because both protein activity and turnover are closely tied to protein post-translational modification (e.g., phosphorylation and ubiquitination), new technologies are required for the large-scale identification, characterization, and quantification of protein post- translational modifications. Reversible protein phosphorylation is a general process affecting most cellular regulatory systems. Phosphorylation is critical to maintaining normal physiology; malfunctions in protein phosphorylation have been implicated in the etiology of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. Protein phosphorylation is an intense research area with great promise for improving human health. Technologies developed under the previous cycle of this grant allowed for the rapid, sensitive and accurate identification of thousands of phosphorylation sites from a single sample. This landmark achievement brings other important unsolved issues to light. Two of the most critical are addressed in this proposal - quantitative phosphorylation profiling and phosphorylation site occupancy. For Aim 1, we will develop quantitative strategies to study dynamic phosphorylation on a near-global scale. This includes combining and integrating a robust stable isotope labeling strategy, new enrichment approaches, and new software tools with our existing platform. For Aim 2, we will perform a highly focused set of experiments to globally profile phosphorylation differences after DNA damage in both yeast cells and human cell lines. These experiments will provide a biologically important proving ground for newly developed techniques and software. Finally, for Aim 3, we will develop and apply a strategy to determine the basal site occupancy levels for all kinases (87) in the tandem affinity purification (TAP) yeast library. In addition, site occupancy levels for a set of human kinases will be examined for DNA damage- and cell cycle-dependency. PUBLIC HEALTH RELEVANCE Protein phosphorylation is an intense research area with great promise for the improvement of human health. In addition to its critical role in normal physiology, malfunctions in protein phosphorylation have been implicated as a contributing factor in the causation of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. This proposal will provide new technologies to profile phosphorylation events between different cell states on a global scale.