Figure 4.

Schematic representation of HDX MS work flow to examine protein stability. The exchange is initiated by placing the unlabeled protein into a D₂O-based solvent system (e.g., by a rapid dilution). Unstructured and highly dynamic protein segments undergo fast exchange (blue and red colors represent protons and deutrons, respectively). Following the quench step (rapid solution acidification and temperature drop), the protein loses its native conformation, but the spatial distribution of backbone amide protons and deuterons across the backbone is preserved (all labile hydrogen atoms at side chains undergo fast back-exchange at this step). Rapid clean-up followed by MS measurement of the protein mass reports the total number of backbone amide hydrogen atoms exchanged under native conditions (a global measure of the protein stability under native conditions), as long as the quench conditions are maintained during the sample work-up and measurement. Alternatively, the protein can by digested under the quench conditions using acid-stable protease(s), and LC/MS analysis of masses of individual proteolytic fragments will provide information on the backbone protection of corresponding protein segments under the native conditions.