Zhou Jian, Lin Yong, Deng Xingcan, Shen Jianying, He Quanyuan, Chen Ping, Wang Xianchun, Liang Songping
College of Life Sciences, Hunan Normal University, Changsha 410081, P. R. China.
J Proteome Res. 2008 Apr;7(4):1778-83. doi: 10.1021/pr070526j. Epub 2008 Feb 29.
Analysis of membrane proteins, particularly integral membrane proteins, still presents a great challenge due to their poor water solubility and low abundance though much effort has been devoted to the solubilization and enrichment of the protein class. In this paper, a two-phase, on-membrane digestion method was developed and applied in the analysis of rat liver membrane proteome. The two-phase system was constituted by mixing n-butanol and 25 mM NH4HCO3. Comparative experiments indicated that the proteins on membranes could be digested in the two-phase system more efficiently than in both 60% methanol and 25 mM NH4HCO3 solutions under the same conditions, thereby improving the identification of the membrane proteins. When the established two-phase system and CapLC-MS/MS was used to analyze rat liver membrane proteome, a total of 411 membrane proteins were identified, more than 80% of which were transmembrane proteins with 1-12 mapped transmembrane domains (TMDs). Because of its extraction and dissolution actions, the two-phase on-membrane digestion system we developed could efficiently improve the digestion and removal of adsorbed nonmembrane proteins, and remarkably increase the number and coverage of identified membrane proteins, particularly the transmembrane proteins. Using our procedure to identify a complementary protein set from all fractions of the two-phase system could achieve a higher coverage of the membrane proteome.
尽管人们在膜蛋白，尤其是整合膜蛋白的增溶和富集方面付出了诸多努力，但由于其水溶性差且丰度低，对其进行分析仍然是一项巨大的挑战。本文开发了一种两相膜上消化方法，并将其应用于大鼠肝脏膜蛋白质组的分析。该两相系统由正丁醇和25 mM碳酸氢铵混合而成。对比实验表明，在相同条件下，膜上的蛋白质在两相系统中比在60%甲醇和25 mM碳酸氢铵溶液中能更有效地被消化，从而提高了膜蛋白的鉴定率。当使用所建立的两相系统和纳升液相色谱-串联质谱（CapLC-MS/MS）分析大鼠肝脏膜蛋白质组时，共鉴定出411种膜蛋白，其中80%以上是具有1至12个跨膜结构域（TMD）的跨膜蛋白。由于其提取和溶解作用，我们开发的两相膜上消化系统能够有效地改善对吸附的非膜蛋白的消化和去除，并显著增加已鉴定膜蛋白，特别是跨膜蛋白的数量和覆盖率。使用我们的方法从两相系统的所有组分中鉴定互补蛋白集，可以实现对膜蛋白质组更高的覆盖率。
