Mass Spectrometry-Based Footprinting Strategies for GPCR Structure Analysis

Abstract G protein-coupled receptors (GPCRs) are a major class of membrane proteins that mediate diverse signaling pathways and represent the largest family of drug targets. However, their conformational flexibility and structural sensitivity to membrane environments make it difficult to characterize using conventional structural methods such as X-ray crystallography, cryo-electron microscopy, and NMR. Mass spectrometry (MS)-based footprinting has been a powerful approach for probing GPCR structure and dynamics. Well-designed footprinting strategies report on solvent accessibility changes with distinct spatial and temporal resolutions. In this review, we discuss how temporal footprinting and native-like membrane mimetics such as liposomes, nanodiscs, picodiscs, and styrene maleic acid lipid particles (SMALPs) have expanded the ability to characterize GPCR dynamics under relevant physiological conditions. We also focus on four representative footprinting techniques, including fast photochemical oxidation of proteins (FPOP), synchrotron X-ray footprinting, carbene labeling, and hydrogen–deuterium exchange (HDX), and discuss the structural information on GPCRs that each method provides. We highlight the differences of each approach when analyzing GPCRs, including labeling chemistry, time scale, residue specificity, and structural resolution. We also summarize their applications in studying GPCR/ligand interaction and conformational transitions. Finally, we discuss current challenges and future opportunities for integrating footprinting techniques to achieve a more comprehensive understanding of the GPCR structure relevant to drug discovery.