Complementary Structural Information for Antibody-Antigen Complexes from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry

Characterizing antibody-antigen interactions is necessary for properly developing therapeutic antibodies, understanding their mechanisms of action, and patenting new drug molecules. Here, we demonstrate that hydrogen-deuterium exchange (HDX) mass spectrometry (MS) measurements together with diethylpyrocarbonhigher order structural information about antibody-antigen interactions that is not available from either technique alone. Using the well-characterized model system of tumor necrosis factor alpha (TNF alpha) in complex with three different monoclonal antibodies (mAbs), we show that two techniques offer a more complete overall picture of TNF alpha's structural changes upon binding different mAbs, sometimes providing synergistic information about binding sites and changes in protein dynamics upon binding. Labeling decreases in CL generally occur near the TNF alpha epitope, whereas decreases in HDX can span the entire protein due to substantial stabilization that occurs when mAbs bind TNF alpha. Considering both data sets together clarifies the TNF alpha regions that undergo a decrease in solvent exposure due to mAb binding and that undergo a change in dynamics due to mAb binding. Moreover, the single-residue level resolution of DEPC-CL/MS can clarify HDX/MS data for long peptides. We feel that the two techniques should be used together when studying the mAb-antigen interactions because of the complementary information they provide.

Automated summary

Characterizing antibody-antigen interactions is crucial for the development of therapeutic antibodies, understanding their mechanisms, and patenting new drug molecules. By combining HDX mass spectrometry measurements with DEPC-CL, more comprehensive information about the structural changes and dynamics of antibody-antigen interactions can be obtained. These two techniques synergistically provide insights into binding sites, protein stabilization, and solvent exposure.