Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D₂O) for a variable ‘labeling time’, resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired. When coupled with MS, HDX is suitable for probing allosteric effects in catalysis and ligand binding, epitope mapping, validation of biosimilars, drug candidate screening and mapping membrane-protein interactions among many other bioanalytical applications. This review introduces HDX-MS via a brief description of HDX-MS development, followed by an overview of HDX theory and ultimately an outline of methods and procedures involved in performing HDX-MS experiments.

大约25年前，氢/氘交换（HDX）质谱（MS）成为一种用于生物化学和结构生物学的工具。此后，它已成为研究蛋白质动力学，蛋白质-配体相互作用，膜蛋白质和固有无序蛋白质（IDP）的关键方法。在HDX标记中，蛋白质暴露于氘化溶剂（通常为D ₂O）在可变的“标记时间”内，导致酰胺主链和氨基酸侧链上未保护的不稳定质子的同位素交换。通过比较蛋白质不同区域中氘的摄取水平，可以获取有关系统构象和动态变化的信息。当与MS结合使用时，HDX适用于在催化和配体结合，表位作图，生物仿制药的验证，候选药物筛选以及膜蛋白相互作用等许多其他生物分析应用中探索变构作用。这篇评论通过对HDX-MS开发的简要介绍来介绍HDX-MS，然后概述了HDX理论，并最终概述了进行HDX-MS实验所涉及的方法和过程。