Electrochemical Generation of Hydroxyl Radicals for Examining Protein Structure
Electrochemical Generation of Hydroxyl Radicals for Examining Protein Structure
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DOI：
10.1021/ac400107c
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发表时间：
2013-07-02
影响因子：
7.4
通讯作者：
Heien, Michael L.
中科院分区：
文献类型：
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作者：
Monroe, Eric B.;Heien, Michael L.
利用羟基自由基共价标记蛋白质暴露在溶剂中的表面，已被证明是研究蛋白质结构和分子间界面的有力工具。目前用于足迹实验的产生羟基自由基的方法依赖于过氧化氢的激光光解或水的同步辐射分解，这大大增加了实验的成本和/或复杂性。在这项工作中，我们开发了电Fenton反应作为一种产生羟基自由基的方法，用于结构足迹质谱学实验，以补充现有的基于激光和同步加速器的方法，同时降低了启动此类实验的成本和复杂性。使用电化学流动电池还能够控制自由基产生过程的时间和程度，同时降低通常与自由基足迹实验相关的复杂性。用Fenton产生的羟基自由基对模型蛋白质泛素进行标记，并用自顶向下的蛋白质组学方法验证蛋白质表面的氧化位点。
The use of hydroxyl radicals to covalently label the solvent-exposed surface of proteins has been shown to be a powerful tool to examine the structure of proteins and intermolecular interfaces. Current methods to generate hydroxyl radicals for footprinting experiments rely on the laser photolysis of H2O2 or the synchrotron radiolysis of water, which adds significant costs and/or complexity to the experiments. In this work, we develop the electro-Fenton reaction as a means to generate hydroxyl radicals for structural footprinting mass spectrometry experiments to complement current laser and synchrotron-based methods, while reducing the costs and complexity of initiating such experiments. The use of an electrochemical flow cell also enables control of the timing and extent of the radical generation process, while reducing the complexity typically associated with radical footprinting experiments. Ubiquitin, a model protein, was labeled with electro-Fenton generated hydroxyl radicals and top down proteomics was used to verify oxidation sites on the protein surface.