Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow.

Analysis of disulfide-bonded proteins by hydrogen/deuterium exchange mass spectrometry (HDX-MS) requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench solution (e.g., tris(2-carboxyethyl)phosphine (TCEP)). The chemical reduction, however, is severely limited under quenched conditions due to a narrow time window as well as low pH and temperature. Here, we demonstrate the real-world applicability of integrating electrochemical reduction into an online HDX-MS workflow. We have optimized the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and nerve growth factor-β (NGF). Several different parameters (flow rate and applied square wave potential, as well as the type of labeling and quench buffer) were investigated, and the ...