Synergistic Structural Information from Covalent Labeling and Hydrogen-Deuterium Exchange Mass Spectrometry for Protein-Ligand Interactions.

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) and covalent labeling (CL) MS are typically considered to be complementary methods for protein structural analysis, because one probes the protein backbone, while the other probes side chains. For protein-ligand interactions, we demonstrate in this work that the two labeling techniques can provide synergistic structural information about protein-ligand binding when reagents like diethylpyrocarbonate (DEPC) are used for CL because of the differences in the reaction rates of DEPC and HDX. Using three model protein-ligand systems, we show that the slower time scale for DEPC labeling makes it only sensitive to changes in solvent accessibility and insensitive to changes in protein structural fluctuations, whereas HDX is sensitive to changes in both solvent accessibility and structural fluctuations. When used together, the two methods more clearly reveal binding sites and ligand-induced changes to structural fluctuations that are distant from the binding site, which is more comprehensive information than either technique alone can provide. We predict that these two methods will find widespread usage together for more deeply understanding protein-ligand interactions.