Kinase Profiling with Quantititative Chemoproteomics

描述（由申请人提供）：本项目的长期目标是开发一种快速定量的方法，用于全面分析肿瘤细胞中的预后信号级联。已知蛋白激酶参与较大的大分子复合物，其将细胞外信号传递到表型反应中。因为这些复合物作为细胞刺激的整合子和效应子，所以激酶的丰度和活性经常被用作细胞状态的报告子。通过选择性地富集含有激酶的信号复合物，我们将能够剖析癌细胞的分子布线。本申请中提出的研究目的是开发和应用一套亲和试剂，其可用于选择性富集活化的信号传导复合物。在我们的第一个目标中，我们将产生靶向含有激酶和磷酸酪氨酸残基的信号复合物的二价亲和试剂。我们的二价亲和试剂将包含Grb 2-SH 2结构域和激酶抑制剂，其应分别特异性结合磷酸酪氨酸残基和激酶ATP结合位点。我们将使用基于SILAC的定量蛋白质组学评估我们的二价抑制剂选择性富集信号复合物而不是其单价组分的能力。这些研究将指导我们对二价抑制剂方法的模块化的理解，并为未来探针的开发提供设计原则。对于我们的第二个目标，我们的目标是设计SH 2激酶抑制剂靶向不同的细胞信号传导途径，并评估其能力，以丰富的途径特异性蛋白质复合物。我们的目标是开发二价亲和试剂，其对组装的蛋白质复合物而不是单一蛋白质具有独特的和靶向的亲和力。我们的应用是使用基于激酶通道的亲和试剂来富集来自 生物样品克服了常规生化分析的动态范围限制。我们的抑制剂-树脂将被设计为富集家族或信号传导蛋白途径中的特定激酶，并与定量MS结合使用以测定激酶丰度和活性。

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.