Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, PR China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jul 15;901:18-24. doi: 10.1016/j.jchromb.2012.05.035. Epub 2012 Jun 4.
Although much effort has been made in the field of membrane proteomics, the analysis of membrane proteins particularly integral membrane proteins with poor water solubility still presents a great challenge. In this paper, 2% SDS was used to extract membrane proteins and experimental conditions for the application of acetone precipitation method to the cleanup of SDS-solubilized membrane protein sample were optimized. For improving the re-dissolution and trypsinolysis of acetone-precipitated proteins, several commonly used additives, urea, methanol and sodium deoxycholate (SDC), were employed and compared. The results showed that, when the pre-cooled acetone-to-sample ratio was 6:1 (v/v) with one additional washing step, residual SDS in the protein sample could be lowered to below 0.01% and more than 90% of the proteins were precipitated and therefore recovered. 1% SDC-containing buffer could improve the re-dissolution and digestion of the acetone precipitated proteins more efficiently than the others. Using the combinative sample preparation strategy developed, 398 proteins were identified from the rat liver membrane-enriched fraction, including 188 membrane proteins. Compared with other three representative solution-based sample preparation methods commonly used in membrane proteomics, the newly developed combinative strategy increased the number of identified total proteins and membrane proteins on average by 29.2% and 28.5%, respectively. This combinative strategy was demonstrated to be easily operated at low cost and suitable for the analysis of membrane proteins varying in type and sample volume, etc.
尽管在膜蛋白质组学领域已经付出了大量努力，但分析膜蛋白质，特别是水溶性差的整合膜蛋白质，仍然是一个巨大的挑战。本文采用 2% SDS 提取膜蛋白，并对丙酮沉淀法用于 SDS 溶解膜蛋白样品的净化条件进行了优化。为了提高丙酮沉淀蛋白的重溶解和胰酶水解效率，比较了几种常用的添加剂，包括尿素、甲醇和脱氧胆酸钠（SDC）。结果表明，当预冷的丙酮与样品的体积比为 6:1（v/v），并增加一次洗涤步骤时，可将蛋白质样品中的 SDS 残留量降低至 0.01%以下，且可沉淀并回收 90%以上的蛋白质。含 1% SDC 的缓冲液可更有效地提高丙酮沉淀蛋白的重溶解和消化效率。采用该联合样品制备策略，从大鼠肝膜富集部分鉴定到 398 种蛋白质，其中包括 188 种膜蛋白。与膜蛋白质组学中常用的另外三种代表性的基于溶液的样品制备方法相比，新开发的联合策略平均分别增加了 29.2%和 28.5%的总蛋白和膜蛋白的鉴定数量。该联合策略操作简单，成本低，适用于分析不同类型和不同样品量的膜蛋白等。
