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.
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