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.

Electrospray ionization (ESI) generates intact gas-phase ions from analytes in solution for mass spectrometric investigations. ESI can proceed via different mechanisms. Low molecular weight analytes follow the ion evaporation model (IEM), whereas the charged residue model (CRM) applies to large globular species. A chain ejection model (CEM) has been proposed for disordered polymers.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

The folding and misfolding mechanism of multi-domain proteins remains poorly understood. While thermodynamic instability of the first nucleotide binding domain (NBD1) of ΔF508-CFTR partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508-CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane spanning domain 2) interface stabilization are required for wild-type-like folding, processing and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508-CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multi-domain membrane proteins.

The acid-induced denaturation of ferricytochrome c (cyt c) was examined in aqueous solutions containing different concentrations of methanol by electrospray ionization mass spectrometry (ESI MS) and optical spectroscopy. Circular dichroism, fluorescence, and absorption spectroscopy show that at a low concentration of methanol (3%) a decrease in pH induces a cooperative unfolding transition at around pH 2.6 that is accompanied by a breakdown of the native secondary and tertiary structure of the protein. In 50% methanol the breakdown of the tertiary structure occurs at around pH 4.0, whereas the alpha-helical content remains largely intact over the whole pH range studied. In ESI MS different protein conformations in solution are monitored by the different charge state distributions they generate during ESI. The ESI mass spectra recorded at near-neutral pH for both methanol concentrations are very similar and show a maximum at (cyt c + 8H+)8+. Despite the different conformations of the protein in solution, the acid-denatured states for the two methanol concentrations also show very similar mass spectra with a maximum at (cyt c + 17H+)17+. This indicates that the charge state distribution generated during ESI is not sensitive to the differences in the secondary structure of the denatured protein. The observed transition from low to high charge states is due to the breakdown of the tertiary structure in both cases. These findings suggest that ESI MS might be a general method to selectively monitor changes in the tertiary structure of proteins.