Kinase Profiling with Quantititative Chemoproteomics

使用定量化学蛋白质组学进行激酶分析

• 批准号: 8546539
• 负责人: Dustin J Maly
• 金额: $ 23.52万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8546539
• 关键词: Affinity; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biomedical Research; Cancer Cell Growth; Cell Proliferation; Cells; Complex; Coupled; Coupling; Development; Diagnostic; Disease; Drug Targeting; Enzymes; Epidermal Growth Factor; Family; Fibroblast Growth Factor; Fingerprint; Future; Goals; Growth Factor; Insulin; Insulin-Like Growth Factor I; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Methodology; Methods; Molecular; Molecular Analysis; Pathway interactions; Phosphotransferases; Phosphotyrosine; Plant Resins; Play; Post-Translational Protein Processing; Protein Kinase; Proteins; Proteomics; Reagent; Reporter; Research; Role; Sampling; Series; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Stimulus; System; Technology; Testing; Ubiquitin; base; cancer cell; design; inhibitor/antagonist; kinase inhibitor; neoplastic cell; novel; novel strategies; prognostic; protein complex; public health relevance; response; src Homology Region 2 Domain

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop a rapid and quantitative method for globally profiling prognostic signaling cascades in tumor cells. Protein kinases are known to participate in larger macromolecular complexes that transmit extra-cellular signals into phenotypic responses. Because these complexes act as integrators and effectors of cellular stimuli, the abundance and activity of kinases are often used as reporters for the cellular state. By selectively enriching kinase-containing signaling complexes, we will be able to dissect the molecular wiring of cancer cells. The objective of the research proposed in this application is to develop and apply a suite of affinity reagents that can be used to selectively enrich activated signaling complexes. In our first aim, we will generate bivalent affinity reagents targeting signaling complexes containing a kinase and phosphotyrosine residues. Our bivalent affinity reagent will comprise a Grb2-SH2 domain and a kinase inhibitor, which should specifically bind to phosphotyrosine residues and kinase ATP-binding sites, respectively. We will evaluate the abilities of our bivalent inhibitors to selectively enrich signaing complexes over its monovalent components using SILAC-based quantitative proteomics. These studies will guide our understanding of the modularity of our bivalent inhibitor approach and provide design principles for development of future probes. For our second aim, our goal is to design SH2-kinase inhibitors targeting distinct cell signaling pathways and evaluate their ability to enrich pathway-specific protein complexes. Our goal is to develop bivalent affinity reagents that have distinct and targeted affinities to assembled protein complexes instead of single proteins. Our application is to use kinase inhibitor-based affinity reagents to enrich kinases from biological samples overcomes the dynamic range limitations of conventional biochemical analyses. Our inhibitor- resins will be designed to enrich specific kinases in families or signalin pathways and used in combination with quantitative MS to assay both kinase abundance and activity.

描述(由申请人提供)：该项目的长期目标是开发一种快速和定量的方法，用于全局描述肿瘤细胞中的预后信号级联。已知蛋白激酶参与更大的大分子复合体，这些复合体将细胞外信号传递为表型反应。因为这些复合体是细胞刺激的整合体和效应器，所以激酶的丰度和活性经常被用来作为细胞状态的报告。通过选择性地富集含激酶的信号复合体，我们将能够剖析癌细胞的分子线路。本申请中提出的研究目的是开发和应用一套亲和试剂，用于选择性地丰富激活的信号复合体。在我们的第一个目标中，我们将产生针对含有一种激酶和磷酸酪氨酸残基的信号复合体的二价亲和试剂。我们的二价亲和试剂将包括一个Grb2-SH2结构域和一个激酶抑制剂，它们应该分别与磷酸酪氨酸残基和激酶ATP结合位点特异性结合。我们将使用基于SILAC的定量蛋白质组学来评估我们的二价抑制剂选择性地丰富其单价组分的信号复合体的能力。这些研究将指导我们对我们的二价抑制剂方法的模块化的理解，并为未来的探针开发提供设计原则。对于我们的第二个目标，我们的目标是设计针对不同细胞信号通路的SH2-激酶抑制剂，并评估它们丰富通路特异性蛋白复合体的能力。我们的目标是开发对组装的蛋白质复合体而不是单一蛋白质具有明显和靶向亲和力的二价亲和试剂。我们的应用是使用基于激酶抑制剂的亲和试剂来丰富来自 生物样品克服了传统生化分析的动态范围限制。我们的抑制树脂将被设计用来丰富家族或信号通路中的特定激酶，并与定量MS结合使用来检测激酶的丰度和活性。