Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134, United States.
Department of Chemistry and Biochemistry and Bio5 Institute, University of Arizona, Tucson, Arizona 85721, United States.
Anal Chem. 2023 Nov 28;95(47):17212-17219. doi: 10.1021/acs.analchem.3c02164. Epub 2023 Nov 14.
Membrane proteins represent the majority of clinical drug targets and are actively involved in a range of cellular processes. However, the complexity of membrane mimetics for membrane protein solubilization poses challenges for native mass spectrometry (MS) analyses. The most common approach for native MS analyses of membrane proteins remains offline buffer exchange into native MS-compatible buffers prior to manual sample loading into static nano-ESI emitters. This laborious process requires relatively high sample consumption and optimization for the individual proteins. Here, we developed online buffer exchange coupled to native mass spectrometry (OBE-nMS) for analyzing membrane proteins in different membrane mimetics, including detergent micelles and nanodiscs. Detergent screening for OBE-nMS reveals that mobile phases containing ammonium acetate with lauryl-dimethylamine oxide are most universal for characterizing both bacterial and mammalian membrane proteins in detergent. Membrane proteins in nanodiscs simply require ammonium acetate as the mobile phase. To preserve the intact nanodiscs, a novel switching electrospray approach was used to capture the high-flow separation on the column with a low-flow injection to MS. Rapid OBE-nMS completes each membrane protein measurement within minutes and thus enables higher-throughput assessment of membrane protein integrity prior to its structural elucidation.
膜蛋白是大多数临床药物靶点的代表，它们积极参与多种细胞过程。然而，膜模拟物对于膜蛋白溶解的复杂性给天然质谱（MS）分析带来了挑战。对于膜蛋白的天然 MS 分析，最常用的方法仍然是在手动将样品加载到静态纳升电喷雾发射器之前，将其在线缓冲交换为天然 MS 兼容的缓冲液。这个繁琐的过程需要相对较高的样品消耗，并针对每个蛋白质进行优化。在这里，我们开发了在线缓冲交换与天然质谱（OBE-nMS）相结合的方法，用于分析不同膜模拟物中的膜蛋白，包括去污剂胶束和纳米盘。用于 OBE-nMS 的去污剂筛选表明，含有月桂基二甲基氧化胺的乙酸铵流动相对于在去污剂中表征细菌和哺乳动物膜蛋白最通用。纳米盘中的膜蛋白只需使用乙酸铵作为流动相。为了保持完整的纳米盘，我们使用了一种新颖的切换电喷雾方法，用低流速注射到 MS 上，在柱上捕获高流速分离。快速的 OBE-nMS 在几分钟内完成每个膜蛋白的测量，从而能够在其结构阐明之前，对膜蛋白的完整性进行更高通量的评估。
