A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry.
A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry.
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DOI：
10.1016/j.ab.2011.07.015
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发表时间：
2011-11-15
影响因子：
2.9
通讯作者：
Hall MC
中科院分区：
文献类型：
--
作者：
Eissler CL;Bremmer SC;Martinez JS;Parker LL;Charbonneau H;Hall MC
大多数真核生物蛋白质在体内被磷酸化，磷酸化可能是最常见的调节性翻译后修饰。许多蛋白质在许多位点被磷酸化，通常被多种激酶磷酸化，这可能具有不同的功能后果。理解磷酸化事件的生物学功能需要检测和量化底物内各个位点的方法。在这里，我们概述了一个通用的策略，解决了这一需求，并依赖于高灵敏度和特异性的选择反应监测（SRM）质谱，使其潜在的有用的研究在体内磷酸化，而不需要分离靶蛋白。我们的方法使用无标记的量化简单和普遍适用性，虽然它是同样兼容的稳定同位素定量方法。我们证明，无标记的SRM为基础的定量是可比的，用于测量磷酸酶和激酶反应的动力学在体外的常规测定。我们还证明了这种方法的能力，同时测量相对速率的磷酸化和去磷酸化的底物混合物，包括在完整的蛋白质底物的背景下的全细胞提取物的个别网站。这种策略对于表征激酶和磷酸酶的生理底物特异性特别有用，并且也可以应用于其他蛋白质修饰的研究。
The majority of eukaryotic proteins are phosphorylated in vivo and phosphorylation may be the most common regulatory post-translational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases, and can be applied to studies of other protein modifications as well.
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影响因子：
4.1
作者：
HARDER, KW;OWEN, P;JIRIK, FR
通讯作者：
JIRIK, FR
影响因子：
10.5
作者：
CohenFix, O;Peters, JM;Koshland, D
通讯作者：
Koshland, D
DOI：
10.1073/pnas.0607084104
发表时间：
2007-02-13
影响因子：
11.1
作者：
Chi, An;Huttenhower, Curtis;Hunt, Donald F.
通讯作者：
Hunt, Donald F.
影响因子：
2.9
作者：
Placzek, Ekaterina A.;Plebanek, Michael P.;Parker, Laurie L.
通讯作者：
Parker, Laurie L.
影响因子：
3.3
作者：
Kramer, ER;Scheuringer, N;Peters, JM
通讯作者：
Peters, JM