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

Macromolecular organization of proteins and lipids in cellular membranes is fundamental to cell functionality. Recent advances in native mass spectrometry (nMS) have established it as a key analytical tool for capturing these associations. This typically necessitates the extraction of target membrane proteins from their physiological environments into detergent-like surroundings. In our recent studies using in vitro synthetic liposomes, we discovered that gas phase supercharging can selectively destabilize lipid bilayers and enable MS1 detection of embedded and associated protein-lipid complexes. Here, we further extend and apply this methodology to native cell-derived membrane vesicles. We demonstrate our ability to detect and ID protein complexes and their proteoforms directly from native membranes using supercharger-assisted pre-quadrupole activation followed by downstream native top-down MS/MS that combines both collision-based and electron capture-based fragmentations approaches. We first demonstrated this approach through native top-down identification of several integral membrane proteins from in vitro membranes. Subsequently, we developed a protocol to produce nMS-ready native membrane vesicles. Applying to E. coli total membranes, we generated nMS-ready vesicles and identified both integral and membrane-associated protein complexes of homomeric and heteromeric nature using our supercharging-enabled nTD-platform. For the hetero-pentameric BAM-complex, which includes the integral membrane protein BAM-A, we detected several lipidated proteoforms. For peripheral homo-dimeric DLDH, we identified bound endogenous metabolite co-factors. Furthermore, using BAM-complex, a crucial antibiotic target, we show how this platform could be utilized to study drug binding to membrane proteins directly from their native membranes.