Figure 1.

Overview of a typical workflow for an epitope/paratope mapping HDX-MS experiment. Separately, the Ag, Ab-Ag complex, and Ab are labeled in D₂O and incubated for varying lengths of time. The reactions are then quenched at low pH and low temperature. The protein samples are digested (typically with pepsin) to generate peptide fragments. Peptide fragments from each sample are analyzed using LC-MS to identify mass differences at various time points. The D uptake altered by binding enables identification of putative paratope and epitope peptides. Figure is adapted from (20).