<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.细胞膜中的蛋白质与脂质对于细胞功能至关重要。原生质谱法（native mass spectrometry, nMS）的技术进展现已可实现对这类复合物的直接分析。此前我们利用合成脂质体开展实验，证实气相超电荷（gas-phase supercharging）可使脂质双层发生去稳定化，从而实现蛋白质-脂质复合物的MS1级检测。本研究将该方法拓展至天然细胞来源囊泡，采用超电荷辅助四极杆前活化（supercharger-assisted pre-quadrupole activation）与自上而下质谱联用（top-down MS/MS）完成蛋白亚型的鉴定。我们通过分析大肠杆菌膜（E. coli membranes）对该方法进行了验证，成功检测到整合膜蛋白及膜相关蛋白复合物，其中包括带有脂化蛋白亚型的BAM复合体（BAM-complex），以及结合了辅因子的DLDH。该技术平台还可用于研究天然环境中药物与膜蛋白的结合相互作用。