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Abstract：
Membrane proteins are underrepresented during proteome characterizations, primarily owing to their lower solubility. Sodium dodecyl sulfate (SDS) is favored to enhance protein solubility but interferes with downstream analysis by mass spectrometry. Here, we present an improved workflow for SDS depletion using transmembrane electrophoresis (TME) while retaining a higher recovery of membrane proteins. Though higher levels of organic solvent lower proteome solubility, we found that the inclusion of 40% methanol provided optimal solubility of membrane proteins, with 86% recovery relative to extraction with SDS. Incorporating 40% methanol during the electrophoretic depletion of SDS by TME also maximized membrane protein recovery. We further report that methanol accelerates the rate of detergent removal, allowing TME to deplete SDS below 100 ppm in under 3 min. This is attributed to a three-fold elevation in the critical micelle concentration (CMC) of SDS in the presence of methanol, combined with a reduction in the SDS to protein binding ratio in methanol (0.3 g SDS/g protein). MS analysis of membrane proteins isolated from the methanol-assisted workflow revealed enhanced proteome detection, particularly for proteins whose pI contributed a minimal net charge and therefore possessed reduced solubility in a purely aqueous solvent. This protocol presents a robust approach for the preparation of membrane proteins by maximizing their solubility in MS-compatible solvents, offering a tool to advance membrane proteome characterization.
摘要：
膜蛋白在蛋白质组表征过程中代表性不足，主要是由于它们的溶解度较低。十二烷基硫酸钠（SDS）有利于提高蛋白质溶解度，但会干扰质谱的下游分析。这里,我们提出了使用跨膜电泳（TME）进行SDS消耗的改进工作流程，同时保留了较高的膜蛋白回收率。虽然较高水平的有机溶剂会降低蛋白质组的溶解度，我们发现包含40%的甲醇提供了膜蛋白的最佳溶解度，相对于SDS提取,回收率为86%。在通过TME电泳消耗SDS期间掺入40%甲醇也使膜蛋白回收率最大化。我们进一步报告，甲醇加速洗涤剂的去除速度，允许TME在3分钟内消耗低于100ppm的SDS。这归因于在甲醇存在下SDS的临界胶束浓度（CMC）提高了三倍，结合在甲醇中SDS与蛋白质结合比的降低(0.3gSDS/g蛋白质)。从甲醇辅助工作流程中分离的膜蛋白的MS分析显示增强的蛋白质组检测，特别是对于其pi贡献最小净电荷并因此在纯水性溶剂中具有降低的溶解度的蛋白质。该协议提出了一种强大的方法，通过最大化它们在MS兼容溶剂中的溶解度来制备膜蛋白，提供了一种工具来推进膜蛋白质组表征。
