Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.
Sensitive multiplexed analysis of kinase activities and activity-based kinase identification.
复制标题
DOI：
10.1038/nbt.1566
复制
发表时间：
2009-10
影响因子：
46.9
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
--
作者：
Kubota, Kazuishi;Anjum, Rana;Yu, Yonghao;Kunz, Ryan C.;Andersen, Jannik N.;Kraus, Manfred;Keilhack, Heike;Nagashima, Kumiko;Krauss, Stefan;Paweletz, Cloud;Hendrickson, Ronald C.;Feldman, Adam S.;Wu, Chin-Lee;Rush, John;Villen, Judit;Gygi, Steven P.
一种或多种激酶信号传导途径的组成性激活是许多癌症的标志。在这里，我们扩展了先前描述的基于质谱的KAYAK方法，同时监测多个信号通路的激酶活性。这种改进的单反应策略在单次质谱运行中定量90种合成肽的磷酸化，与纳克至微克量的细胞裂解物相容。此外，该方法通过底物竞争效应增强激酶单特异性，忠实地报告了有丝分裂原刺激后许多信号传导途径的特征或跨一组充分研究的癌细胞系的基础途径活化差异。来自相关实验的活性的层次聚类将由相似激酶磷酸化的肽分组在一起，并且当与使用药理学抑制剂的途径改变组合时，区分效力、脱靶效应和遗传背景的潜在差异。最后，我们介绍了一种策略，以确定激酶，甚至相关的蛋白质复合物的成员，负责感兴趣的磷酸化事件。
Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry–based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.
登录
查看更多内容
影响因子：
64.5
作者：
Foster, LJ;de Hoog, CL;Mann, M
通讯作者：
Mann, M
DOI：
10.1016/0006-291x(74)90234-4
发表时间：
1974-01-01
影响因子：
3.1
作者：
DAILE, P;CARNEGIE, PR
通讯作者：
CARNEGIE, PR
影响因子：
64.8
作者：
Andersen, JS;Wilkinson, CJ;Mann, M
通讯作者：
Mann, M
影响因子：
7
作者：
Janes, KA;Albeck, JG;Yaffe, MB
通讯作者：
Yaffe, MB
影响因子：
4.8
作者：
Diks, SH;Kok, K;Peppelenbosch, MP
通讯作者：
Peppelenbosch, MP