Native Top-down analysis of membrane protein complexes directly from in vitro and native membranes

Proteins and lipids in cellular membranes are vital for cell function. Advances in native mass spectrometry (nMS) now allow direct analysis of these complexes. Previously, we used synthetic liposomes to show that gas-phase supercharging destabilizes lipid bilayers, enabling MS1 detection of protein-lipid complexes.Here, we extend this method to native cell-derived vesicles, using supercharger-assisted pre-quadrupole activation and top-down MS/MS for proteoform identification. We validated this approach by analyzing E. coli membranes and detecting integral and membrane-associated protein complexes, including the BAM-complex (with lipidated proteoforms) and DLDH (with bound cofactors). This platform also enables studying drug binding to membrane proteins in their native environments.