Mass spectrometry and chemical footprinting for probing protein interfaces

Chemical modifications have been developed over the years to footprint proteins, to determine interfaces and sites of ligand interaction, and to understand protein folding. We are implementing these procedures in protein mass spectrometry. In H/D amide exchange, we use our approach of PLIMSTEX (Protein Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) to determine binding affinity and to locate binding regions between a protein and ligand. Complementing H/DX are highly specific reactions including that with glycyl ethyl ester (GEE) as footprinting procedures. The latter can determine the properties of proteins in complex media (e.g., in photosynthetic bacteria or membranes). An intermediate strategy is OH radical footprinting, which labels aromatic, some aliphatic, and sulfur‐containing amino acids in proteins (termed FPOP, Fast Photochemical Oxidation of Proteins). FPOP is indeed faster than the fastest early steps of protein unfolding and can be used in a “pump‐probe” experiment to map folding/unfolding. Another tool is Electron Capture Dissociation (ECD), which may be vital in learning about protein assemblies in the gas phase. These chemical approaches are enabled by the powerful analytical proteomic strategies of modern mass spectrometry.