Fig. 2: The common ‘bottom-up’ or ‘local’ HDX-MS experiment.

Proteins are incubated in deuterated buffer for a number of time points, allowing for the incorporation of deuterium into the protein backbone. The exchange reaction is quenched by a shift to acidic pH and a temperature drop (with the optional inclusion of denaturants and reducing agents to enhance protein unfolding). Proteins are then digested by an acid-functional protease, such as pepsin. The proteolytic peptides are desalted and separated using a chilled reversed-phase UHPLC system and eluted into a mass spectrometer, where they are ionized by electrospray and subjected to mass analysis to determine the increase in mass resulting from deuterium uptake. During spectral analysis, the isotopic envelopes of peptides are visualized, and levels of deuteration are determined, typically through comparison of the average mass from the intensity-weighted centroid m/z value (arrows) of the peptide. The example mass spectra show that the peptide has a deuterium level of 2.7 D. The deuterium uptake, resolved to individual peptide segments, is plotted across multiple time points. Peptide uptake plots reveal the local HDX profile of individual protein regions. Peptide uptake plots obtained in an identical manner for multiple states of the protein, such as folded and unfolded, or bound and unbound to a ligand, can be overlaid to enable quick comparison and detection of local differences in HDX (and conformation) between protein states. Such differences in HDX can then be mapped on a three-dimensional representation of the protein to facilitate structural interpretation. Structure adapted from Lee et al.⁵⁸.