Gaoyuan Lu et. al, Sub-residue Resolution Footprinting of Ligand-Protein Interactions by Carbene Chemistry and Ion Mobility-Mass Spectrometry, ACSPublicaitons https://doi.org/10.1021/acs.analchem.9b03827

The knowledge of ligand-protein interactions is essential for understanding fundamental biological processes and for the rational design of drugs that target such processes. Carbene footprinting efficiently labels proteinaceous residues, and has been used with mass spectrometry (MS) to map ligand-protein interactions. Nevertheless, previous footprinting studies are typically performed at residue-level, and therefore the resolution may not be high enough to couple with conventional crystallography techniques. Herein we developed a sub-residue footprinting strategy based on the discovery that carbene labeling produces sub-residue peptide isomers and the intensity changes of these isomers in response to ligand binding can be exploited to delineate ligand-protein topography at sub-residue level. The established workflow combines carbene footprinting, extended liquid chromatographic separation and ion mobility (IM)-MS for efficient separation and identification of sub-residue isomers. Analysis of representative sub-residue isomers located within the binding cleft of lysozyme and those produced from an amyloid-beta segment have both uncovered structural information heretofore unavailable by residue-level footprinting. Lastly, a “real-world” application shows that the reactivity changes of sub-residue isomers at Phe399 can identify the interactive nuances between estrogen-related receptor α, a potential drug target for cancer and metabolic diseases, with its three ligands. These findings have significant implications for drug design. Taken together, we envision the sub-residue level resolution enabled by IM-MS-coupled carbene footprinting can bridge the gap between structural MS and the more-established biophysical tools, and ultimately facilitate diverse applications for fundamental research and pharmaceutical development.