Capillary Zone Electrophoresis-Mass Spectrometry of Intact G Protein-Coupled Receptors Enables Proteoform Profiling

G protein-coupled receptors (GPCRs) are the largest class of integral membrane receptors and responsible for transmitting diverse signals in response to extracellular stimuli. Post-translational modifications serve to dictate the subcellular trafficking and function of a GPCR across space and time. Despite significant interest in mapping the diversity of GPCR modification states (proteoforms), technical challenges have hindered this characterization. While advancements in membrane mimetics and mass spectrometry instrumentation have improved analysis, current workflows require large amounts of homogenous protein, limiting the study of many GPCRs from mammalian sources. Here, we present capillary zone electrophoresis (CZE) as an inline separatory tool for characterizing proteoforms of both intact and partially digested GPCRs. This method allowed for the characterization of multiple proteoforms of both the beta-2-adrenergic receptor and metabotropic glutamate receptor 2 using low sample volumes and without buffer optimization. Notably, in the case of smaller phosphorylated analytes, CZE was able to readily separate positional phosphorylation isomers, and provide superior fragmentation coverage to conventional reverse phase-liquid chromatography (RP-LC).