CHEMICAL GENETIC IDENTIFICATION OF DIRECT KINASE SUBSTRATES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 蛋白质磷酸化是蛋白质翻译后修饰的主要机制，用于控制细胞信号传导。磷蛋白质组学的一个挑战是确定每个蛋白激酶的直接底物。我们已经开发了一种化学策略，将生物正交亲和标签传递到单个蛋白激酶的底物上。目的蛋白激酶被设计成将硫代磷酸部分转移到直接底物上的磷受体羟基上。在第二个非酶步骤中，引入的硫代磷与对硝基甲苯(PNBM)烷基化。针对修饰的硫代磷酸表位的抗体识别这些标记的底物，但不识别其他细胞亲核试剂的烷基化产物。免疫亲和层析可以纯化这些底物，而质谱学为它们的快速鉴定提供了一种有吸引力的方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signalling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. We have developed a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second non-enzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the modified phosphorothioate epitope recognize these labelled substrates, but not alkylation products of other cellular nucleophiles. Immunoaffinity chromatography allows the purification of these substrates, and mass spectrometry provides an attractive method for their rapid identification.