Iranpour Tania, Mirimba Mapenzi, Shenouda Chloe, Lynch Adam, Doucette Alan A
Department of Chemistry, Dalhousie University, 6243 Alumni Crescent, Halifax, NS B3H 4R2, Canada.
Proteomes. 2025 Jul 2;13(3):30. doi: 10.3390/proteomes13030030.
Membrane proteins are preferentially solubilized with sodium dodecyl sulfate (SDS), which necessitates a purification protocol to deplete the surfactant prior to mass spectrometry analysis. However, maintaining solubility of intact membrane proteins is challenged in an SDS-free environment. SDS precipitation with potassium salts (KCl) offers a potentially viable workflow to deplete SDS and permit proteoform analysis. The purpose of this study is to devise a robust detergent-based protocol applicable for processing and analysis of intact membrane-associated proteoforms.
The precipitation conditions impacting SDS removal from spinach chloroplasts and liver membrane proteome preparations were evaluated, capitalizing on optimization of pH (highly basic), addition of MS-compatible solubilizing additives (urea) and adjustment of the KCl to SDS ratio to maximize recovery and purity.
Characterization of the SDS-solubilized, KCl-precipitated spinach membrane preparation revealed multiple charge envelope MS spectra displaying high signal to noise, free of SDS adducts. Precipitation at pH 12 or with urea improved protein recovery and purity. Bottom-up analysis identified 1826 distinct liver protein groups from four independent SDS precipitation conditions. While precipitation at pH 8 without urea revealed a greater number of protein identifications by mass spectrometry, precipitation under highly basic conditions (pH 12) with urea provided higher membrane protein recovery and achieved the greatest number (732 of 1056) and largest percentage (69.3%) of membrane proteins identified in the SDS removal workflow.
This workflow provides new opportunities for MS-based proteoform analysis by capitalizing on the benefits of SDS for protein extraction while maintaining high solubility and purity of intact proteins though KCl precipitation of the surfactant.
膜蛋白优先用十二烷基硫酸钠（SDS）溶解，这就需要一种纯化方案，以便在质谱分析之前去除表面活性剂。然而，在无SDS的环境中维持完整膜蛋白的溶解性具有挑战性。用钾盐（KCl）沉淀SDS提供了一种潜在可行的工作流程，以去除SDS并允许进行蛋白变体分析。本研究的目的是设计一种基于去污剂的稳健方案，适用于完整膜相关蛋白变体的处理和分析。
利用pH值（高碱性）的优化、添加与质谱兼容的增溶添加剂（尿素）以及调整KCl与SDS的比例，评估影响从菠菜叶绿体和肝膜蛋白质组制剂中去除SDS的沉淀条件，以最大限度地提高回收率和纯度。
对SDS溶解、KCl沉淀的菠菜膜制剂的表征显示，多个电荷包络质谱图显示出高信噪比，且无SDS加合物。在pH 12或使用尿素沉淀可提高蛋白质回收率和纯度。自下而上的分析从四个独立的SDS沉淀条件中鉴定出1826个不同的肝蛋白组。虽然在不使用尿素的pH 8条件下沉淀通过质谱鉴定出的蛋白质数量更多，但在高碱性条件（pH 12）下使用尿素沉淀可提供更高的膜蛋白回收率，并在SDS去除工作流程中鉴定出的膜蛋白数量最多（1056个中的732个），所占百分比最大（69.3%）。
该工作流程利用SDS进行蛋白质提取的优势，同时通过表面活性剂的KCl沉淀维持完整蛋白质的高溶解性和纯度，为基于质谱的蛋白变体分析提供了新机会。
