CHEMICAL GENETIC IDENTIFICATION OF DIRECT KINASE SUBSTRATES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 蛋白质磷酸化是用于控制细胞信号传导的翻译后蛋白质修饰的主要机制。 磷酸化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，用于将生物正交亲和标签递送到单个蛋白激酶的底物。 所关注的激酶经工程改造以将硫代磷酸酯部分转移至直接底物上的磷酸受体羟基。 在第二非酶促步骤中，引入的硫代磷酸酯用甲磺酸对硝基苄酯（PNBM）烷基化。 针对修饰的硫代磷酸酯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。 免疫亲和层析允许这些底物的纯化，质谱法提供了一种有吸引力的方法，用于其快速鉴定。

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.