CHEMICAL GENETIC IDENTIFICATION OF DIRECT KINASE SUBSTRATES

直接激酶底物的化学遗传学鉴定

• 批准号: 8169755
• 负责人: KEVAN M. SHOKAT
• 金额: $ 0.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169755
• 关键词: Affinity; Alkylation; Antibodies; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Engineering; Epitopes; Funding; Grant; Individual; Institution; Label; Mass Spectrum Analysis; Methods; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Kinase; Research; Research Personnel; Resources; Signal Transduction; Source; United States National Institutes of Health; chemical genetics; hydroxyl group; immunoaffinity chromatography; interest; phosphorothioate

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 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。 磷酸蛋白质组学的挑战是鉴定每种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力标签递送到单个蛋白激酶的底物上。 感兴趣的激酶旨在将磷酸硫酸盐部分转移到直接底物上的磷酸acceptor羟基。 在第二个非酶步骤中，引入的磷酸硫酸盐用P-硝基苯基甲酯（PNBM）烷基化。 针对改良的磷酸硫酸盐表位的抗体识别这些标记的底物，但不能识别其他细胞亲核菌的烷基化产物。 免疫亲和力色谱允许纯化这些底物，质谱法为它们的快速鉴定提供了一种有吸引力的方法。