Top-Down Analysis of In-Source HDX of Native Protein Ions

Hydrogen/deuterium exchange (HDX) is used in protein biophysics to probe folding dynamics, intermolecular interactions, epitope and other mapping. A typical procedure often involves HDX in buffered D₂O solution followed by pepsin digestion, and liquid chromatography/electrospray ionization mass spectrometry analysis. In this work, HDX of protein ions was conducted in the ESI source. Both native electrospray droplets of ubiquitin and denatured myoglobin were exposed to D₂O vapor in the source region of a Bruker SolariX 12T FTICR-mass spectrometer. Electron capture dissociation was used to assess deuterium incorporation at the residue level. This in-source HDX, on the millisecond-time scale, exchanges side-chain hydrogens and fast-exchanging amides compared to conventional minutes-to-hours HDX of backbone hydrogens in solution with less sample preparation (i.e., no D₂O/protein mixing and incubation, no quenching, protein digestion, or LC separation).