<b>Native Top-down analysis of membrane protein complexes directly from </b><b><i>in vitro</i></b><b> and native membranes</b>

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