Department of Chemistry, The University of Western Ontario, London, ON N6A 5B7, Canada.
Chem Soc Rev. 2011 Mar;40(3):1224-34. doi: 10.1039/c0cs00113a. Epub 2010 Dec 21.
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a number of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissociation methods provide a solution to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful reference for practitioners, who require an overview of some recent trends in HDX/MS.
氘代/氕交换（HDX）质谱（MS）已成为监测溶液中蛋白质结构和动态方面的关键技术。这种方法依赖于这样一个事实，即蛋白质暴露于 D2O 会导致无稳定氢键的无序区域中酰胺 H→D 的快速交换。紧密折叠的元件受到 HDX 的保护程度要高得多，导致由蛋白质的结构动力学（“呼吸运动”）介导的缓慢同位素交换。基于 MS 的肽图谱是测量单个蛋白质片段质量位移的成熟技术。本教程综述简要讨论了 HDX/MS 的基本原理，然后重点介绍了一些最新的发展和应用。与传统的基于蛋白酶解的方法相比，气相碎裂策略代表了一种很有前途的替代方法，但实验人员必须意识到在某些条件下可能会遇到的混乱现象。基于电子的解离方法为解决此问题提供了一种解决方案。我们还讨论了最近的进展，这些进展促进了 HDX/MS 在膜蛋白以及短寿命蛋白质折叠中间体的表征中的应用。希望本综述能为新手提供一个起点，也能为需要了解 HDX/MS 一些最新趋势的从业者提供有用的参考。
