Hydrogen exchange mass spectrometry for studying protein structure and dynamics

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.