Chi Yong, Clurman Bruce E
Divisions of Clinical Research and Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington.
Curr Protoc Chem Biol. 2010 Dec 1;2(4):219-34. doi: 10.1002/9780470559277.ch100151.
Protein kinases constitute a large enzyme family with key roles in cellular signal transduction. One way to elucidate the functions of protein kinases is to systematically identify their downstream targets. Presented here is a simple and effective method to identify direct protein kinase substrates in native cell lysates. First, the activity of the kinase of interest is isolated by engineering the normal kinase to utilize bulky ATP analogs that cannot be used by normal cellular kinases. This allows specific labeling of substrates with thiophosphate tags by performing kinase reactions in cell lysates that also include bulky ATP-γ-S analogs. After digesting the proteins in the reaction mixture, thiophosphopeptides are isolated using a single-step capture-and-release protocol and identified by mass spectrometry. This technique is easy to use and generally applicable. Curr. Protoc. Chem. Biol. 2:219-234 © 2010 by John Wiley & Sons, Inc.
蛋白激酶构成了一个在细胞信号转导中起关键作用的大型酶家族。阐明蛋白激酶功能的一种方法是系统地鉴定其下游靶点。本文介绍了一种在天然细胞裂解物中鉴定直接蛋白激酶底物的简单有效方法。首先，通过改造正常激酶以利用正常细胞激酶无法使用的大体积ATP类似物来分离感兴趣的激酶的活性。这使得通过在还包含大体积ATP-γ-S类似物的细胞裂解物中进行激酶反应，用硫代磷酸标签对底物进行特异性标记。在消化反应混合物中的蛋白质后，使用单步捕获和释放方案分离硫代磷酸肽，并通过质谱鉴定。该技术易于使用且普遍适用。《化学与生物学实验指南》2:219 - 234 © 2010 约翰威立国际出版公司
