Recent applications of mass spectrometry (MS) to study membrane protein complexes are yielding valuable insights into the binding of lipids and their structural and functional roles. To date, most native MS experiments with membrane proteins are based on detergent solubilization. Many insights into the structure and function of membrane proteins have been obtained using detergents; however, these can promote local lipid rearrangement and can cause fluctuations in the oligomeric state of protein complexes. To overcome these problems, we developed a method that does not use detergents or other chemicals. Here we report a detailed protocol that enables direct ejection of protein complexes from membranes for analysis by native MS. Briefly, lipid vesicles are prepared directly from membranes of different sources and subjected to sonication pulses. The resulting destabilized vesicles are concentrated, introduced into a mass spectrometer and ionized. The mass of the observed protein complexes is determined and this information, in conjunction with 'omics'-based strategies, is used to determine subunit stoichiometry as well as cofactor and lipid binding. Within this protocol, we expand the applications of the method to include peripheral membrane proteins of the S-layer and amyloid protein export machineries overexpressed in membranes from which the most abundant components have been removed. The described experimental procedure takes approximately 3 d from preparation to MS. The time required for data analysis depends on the complexity of the protein assemblies embedded in the membrane under investigation.

中文翻译：

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使用超声处理的脂质囊泡进行膜蛋白复合物的质谱分析。

最近应用质谱 (MS) 研究膜蛋白复合物，为了解脂质的结合及其结构和功能作用提供了宝贵的见解。迄今为止，大多数膜蛋白的天然 MS 实验都是基于去污剂溶解。使用去垢剂已经获得了对膜蛋白结构和功能的许多见解；然而，这些可以促进局部脂质重排，并可能导致蛋白质复合物寡聚状态的波动。为了克服这些问题，我们开发了一种不使用清洁剂或其他化学品的方法。在这里，我们报告了一个详细的方案，该方案能够直接从膜上喷射蛋白质复合物，以便通过本机 MS 进行分析。简而言之，直接从不同来源的膜制备脂质囊泡并进行超声脉冲处理。将所得不稳定囊泡浓缩，引入质谱仪并电离。确定观察到的蛋白质复合物的质量，并将该信息与基于“组学”的策略结合起来，用于确定亚基化学计量以及辅因子和脂质结合。在此协议中，我们扩展了该方法的应用，包括 S 层的外周膜蛋白和淀粉样蛋白输出机制，这些机制在膜中过度表达，其中最丰富的成分已被去除。所述实验程序从准备到 MS 大约需要 3 d。数据分析所需的时间取决于所研究的膜中嵌入的蛋白质组装体的复杂性。