New Proteomic Technologies for the Analysis of Tyrosine Kinase Signaling Pathways

描述(由申请人提供)：磷蛋白质组学分析的一个关键目标是深入了解细胞中的蛋白质磷酸化和信号网络。然而，尽管在过去的几年里已经取得了很大的进步，但我们分析这种信号通路的能力仍然受到一些严重的技术挑战的极大限制。长期目标是开发新的蛋白质组学策略，有助于在分子水平上理解磷酸化在调节细胞增殖中的作用。本应用的目的是设计和优化有效和高效的方法和策略，以双功能酪氨酸激酶Syk为模型系统分析酪氨酸激酶磷酸化蛋白质组。这种方法将利用功能性的、可溶的聚合物来高效和包含性地分离磷酸肽，并结合综合的化学、质谱学和计算策略来鉴定特定的酪氨酸激酶底物和定量磷酸蛋白质组学。我们将专注于Syk依赖的信号转导，并建立协议，最终将提供一种强大的方法来剖析任何由蛋白质酪氨酸磷酸化调控的信号通路。在强大的初步数据的指导下，这一目标将通过追求三个具体目标来实现：1)建立一个分析蛋白质磷酸化的蛋白质组学平台。我们将主要开发、表征和优化为磷酸肽的亲和力和化学分离而设计的功能化可溶聚合物；2)鉴定特定的酪氨酸激酶底物及其磷酸化位置；以及3)深入分析几种癌症类型的酪氨酸激酶磷酸化蛋白质组。我们将开发和应用我们的定量蛋白质组学技术来识别和量化几种癌症类型中的酪氨酸磷酸化位点。该项目具有创新性，因为它将开发一系列新的分析技术来直接识别新的酪氨酸激酶底物和磷酸化位点，这将为研究人员提供显著扩展的剖析信号通路的能力。发展的技术将在分子水平上提供详细的知识，确切地说明酪氨酸激酶是如何通过蛋白质磷酸化的动态变化在不同的生理条件下参与细胞信号传递的。人们还预计，通过确定一种激酶的直接底物，这项拟议的研究将提供一种强大的工具，方便地绘制激酶网络图。 公共卫生相关性：拟议的开发一系列分析技术的研究对分子信号领域的研究人员具有潜在的适用性。这些工具将使他们能够研究酪氨酸激酶在多个信号通路中的重要作用。这项拟议的研究将提供独特的策略和材料，加强蛋白质组学对我们理解癌症生物学的重要贡献。

DESCRIPTION (provided by applicant): A key objective of phosphoproteomic analyses is to obtain an in-depth understanding of protein phosphorylation and signaling networks in cells. However, despite the great progress that has been made over the past few years, our ability to analyze such signaling pathways is still greatly limited by a number of serious technical challenges. The long term goal is to develop novel proteomics strategies valuable for understanding on the molecular level the role of phosphorylation in regulating cell proliferation. The objective in this application is to devise and optimize effective and efficient methodologies and strategies for the analyses of tyrosine kinase phosphoproteomes, using a dual functional tyrosine kinase Syk as the model system. This approach will utilize functional, soluble polymers for the efficient and inclusive isolation of phosphopeptides coupled with an integrated chemical, mass spectrometric, and computational strategy for the identification of specific tyrosine kinase substrates and for quantitative phosphoproteomics. We will focus on Syk-dependent signaling and establish protocols that will ultimately provide a powerful method to dissect any signaling pathway regulated by protein-tyrosine phosphorylation. Guided by strong preliminary data, this objective will be achieved by pursuing three specific aims: 1) Develop a proteomic platform for the analysis of protein phosphorylation. We will primarily develop, characterize and optimize functionalized soluble polymers designed for the affinity and chemical isolation of phosphopeptides; 2) Identification of specific tyrosine-kinase substrates and their phosphorylation sites; and 3) In-depth analyses of tyrosine kinase phosphoproteomes in several cancer types. We will develop and apply our quantitative proteomic technologies for the identification and quantification of tyrosine-phosphorylation sites in several cancer types. This project is innovative because it will develop a series of novel analytical techniques to directly identify novel tyrosine kinase substrates and phosphorylation sites that will provide investigators with a significantly expanded capability to dissect signaling pathways. The developed techniques will provide detailed knowledge at the molecular level on precisely how tyrosine kinases are involved in cell signaling under different physiological conditions through dynamic changes in protein phosphorylation. It is also expected that, by identifying a kinase's direct substrates, the proposed research will provide a powerful tool to conveniently map kinase networks. PUBLIC HEALTH RELEVANCE: The proposed studies to develop a series of analytical techniques have potential applicability for investigators in the molecular signaling field. The tools will allow them to examine important roles of tyrosine kinase in multiple signaling pathways. The proposed research will provide unique strategies and materials that will strengthen the important contribution that proteomics can make to our understanding of cancer biology.