Said Hammam H, Doucette Alan A
Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS B3H 4R2, Canada.
Proteomes. 2024 Feb 2;12(1):5. doi: 10.3390/proteomes12010005.
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.
在蛋白质组表征过程中，膜蛋白的代表性不足，主要是因为它们的溶解度较低。十二烷基硫酸钠（SDS）有助于提高蛋白质溶解度，但会干扰质谱的下游分析。在此，我们提出了一种改进的工作流程，通过跨膜电泳（TME）去除SDS，同时保持较高的膜蛋白回收率。尽管较高水平的有机溶剂会降低蛋白质组的溶解度，但我们发现加入40%的甲醇可使膜蛋白的溶解度达到最佳，相对于用SDS提取时，回收率为86%。在通过TME进行SDS的电泳去除过程中加入40%的甲醇，也能使膜蛋白回收率最大化。我们进一步报告称，甲醇可加速去污剂的去除速度，使TME在3分钟内将SDS浓度降至100 ppm以下。这归因于在甲醇存在下SDS的临界胶束浓度（CMC）提高了三倍，同时甲醇中SDS与蛋白质的结合比降低（0.3 g SDS/g蛋白质）。对从甲醇辅助工作流程中分离出的膜蛋白进行质谱分析，结果显示蛋白质组检测得到增强，特别是对于那些pI贡献最小净电荷、因此在纯水溶液中溶解度降低的蛋白质。该方案通过最大化膜蛋白在与质谱兼容的溶剂中的溶解度，为膜蛋白的制备提供了一种可靠的方法，为推进膜蛋白质组表征提供了一种工具。
