A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity
A quantitative mass spectrometry-based approach for identifying protein kinase clients and quantifying kinase activity
复制标题
DOI：
10.1016/j.ab.2010.03.028
复制
发表时间：
2010-07-01
影响因子：
2.9
通讯作者：
Thelen, Jay J.
中科院分区：
文献类型：
--
作者：
Huang, Yadong;Houston, Norma L.;Thelen, Jay J.
据信，智人和拟南芥的基因组分别编码了500多个和1000多个蛋白激酶。尽管如此，很少有真正的激酶-客户关系被详细描述。在这里，我们描述了一种定量质谱（MS）为基础的方法来鉴定激酶客户蛋白。在方法开发过程中，我们使用专用的丙酮酸脱氢酶激酶（PDK）进行体外检测。作为激酶底物，我们使用了合成肽鸡尾酒，并在此过程中证明了该检测既敏感又特异性。一旦检测到肽底物，该方法也可用于表征蛋白激酶-底物动力学。采用无标记光谱计数法，利用丙酮酸脱氢酶E1 α亚基序列衍生的肽底物YHGH(292)SMSDPGSTYR测定PDK的活性。通过研究Met氧化对肽磷酸化的负面影响，进一步验证了光谱计数的效用。我们还测量了不相关的钙依赖性蛋白激酶3 （CPK3）的活性，证明了该方法在蛋白激酶筛选应用中的实用性。(C) 2010爱思唯尔公司版权所有。
The Homo sapiens and Arabidopsis thaliana genomes are believed to encode more than 500 and 1000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Here we describe a quantitative mass spectrometry (MS)-based approach for identifying kinase-client proteins. During method development, we used the dedicated kinase pyruvate dehydrogenase kinase (PDK) for the in vitro assays. As kinase substrate, we used synthetic peptide cocktails and, in the process, demonstrated that the assay is both sensitive and specific. The method is also useful for characterizing protein kinase-substrate kinetics once the peptide substrate is detected. Applying a label-free spectral counting method, the activity of PDK was determined using the peptide substrate YHGH(292)SMSDPGSTYR derived from the pyruvate dehydrogenase E1 alpha subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We also measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications. (C) 2010 Elsevier Inc. All rights reserved.