Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis - Implications for mitochondrial redox signaling
Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis - Implications for mitochondrial redox signaling
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DOI：
10.1074/jbc.m703591200
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发表时间：
2007-07-27
影响因子：
4.8
通讯作者：
Murphy, Michael P.
中科院分区：
文献类型：
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作者：
Hurd, Thomas R.;Prime, Tracy A.;Murphy, Michael P.
由线粒体呼吸链产生的活性氧（ROS）可以是氧化还原信号，但它们是否影响线粒体功能尚不清楚。在这里，我们表明，低水平的ROS从呼吸链在生理条件下可逆地修改硫醇氧化还原状态的线粒体蛋白质参与脂肪酸和碳水化合物代谢。由于这些巯基修饰是特异性的，并且在没有大量巯基变化的情况下发生，我们首先必须开发一种灵敏的技术来鉴定少量被内源性ROS修饰的蛋白质。在这种技术中，氧化还原差异凝胶电泳，控制，和氧化还原挑战的样品标记有不同的硫醇反应性荧光标签，然后在同一个二维凝胶分离，使灵敏的检测硫醇氧化还原修饰的变化，在一个单一的蛋白质斑点内的两个标签的相对荧光，然后通过质谱法的蛋白质鉴定。巯基氧化还原修饰影响酶的活性，这表明来自呼吸链的ROS对酶活性的可逆修饰可能是线粒体氧化还原信号传导的重要且未探索的模式。
Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.