Photodissociation Mass Spectrometry Accurately Localizes Sites of Backbone Deuteration in Peptides

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now positioned as a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to localize incorporated deuterium with single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation to increase the spatial resolution of the HDX-MS experiment, combining high fragmentation efficiency, high fragment ion diversity, and low charge-state dependency.