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首页> 外文期刊>Journal of mass spectrometry: JMS >Future directions of structural mass spectrometry using hydroxyl radical footprinting
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Future directions of structural mass spectrometry using hydroxyl radical footprinting

机译：使用羟基自由基足迹的结构质谱的未来方向

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Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis-driven structural mass spectrometry experiments.
机译：在过去的十年中，已开发了与质谱联用的羟基自由基蛋白质印迹法，并已发展成为一种用于分析蛋白质结构和动力学的强大方法。它已成功应用于蛋白质结构，蛋白质折叠，蛋白质动力学以及蛋白质-蛋白质和蛋白质-DNA相互作用的分析。使用同步加速器辐射分解，蛋白质在“白色” X射线束中的暴露时间为毫秒，可对表面氨基酸侧链提供足够的氧化修饰，可通过质谱轻松检测和定量。因此，可以使用时间分辨方法检查蛋白质或蛋白质复合物中的构象变化，这将是研究大分子动力学的有价值的方法。在这篇综述中，我们描述了羟基自由基蛋白质足迹的新应用，以探测凝溶胶蛋白在毫秒时间尺度上钙依赖性构象变化的时间演变。数据表明，由于不同分子亚结构域中的多个位点具有相似的构象变化速率，因此发生了协同过渡。这些发现表明，时间分辨的蛋白质足迹适合研究从几毫秒到几秒的一段时间内发生的蛋白质动力学。在这篇评论中，我们还展示了如何也可以提高技术的结构分辨率和灵敏度。羟基自由基对不同侧链的反应性变化超过两个数量级，因此在这种实验中很少观察到较低反应性的氨基酸侧链的氧化。在这里，我们证明了基于选择的反应监测（SRM）的方法可用于定量氧化物质，从而提高信噪比。较低反应性的氧化残基组的这种扩展将改善该技术的整体结构分辨率。还建议将此方法作为开发假设驱动的结构质谱实验的基础。

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