茅苍术质膜蛋白的制备及其双向电泳蛋白质组学平台的构建

Establishment of Membrane Proteomics Platform with Twodimensional Electrophoresis for Preparing Identifying Plasma Membrane Proteins from Atractylodes lancea

探讨濒危药用植物茅苍术的质膜蛋白质组方法,旨在达到了解其次级代谢物的代谢途径,并对相关药用活性成分进行代谢调控的目的。以茅苍术的根、茎、叶为实验材料,确定了超速离心结合双水相法作为提取、纯化其质膜蛋白的方法 ;进而对这些质膜蛋白的双向电泳分离条件进行了系统的优化和后期的部分蛋白质鉴定。结果表明,分别采用质量分数为6.4%、6.3%和6.1%的聚合物葡聚糖T-500和聚乙二醇PEG 3350(W/W)的双水相体系对超速离心后的茅苍术根、茎、叶粗膜组分进行处理可获得对应纯度高达92.1%、91.5%和90.8%的质膜蛋白;以2%CHAPS和2%Triton X-100作为组合变性剂裂解膜蛋白,等电聚焦总量为80 000 Vhs,浓度为12.5%-15%的梯度SDS-PAGE分离胶对100μg质膜蛋白进行双向电泳,通过Image Master 2D Platinum 7.0分析软件分别在根、茎、叶的质膜蛋白凝胶图谱上识别出了267、297和248个蛋白斑点;进一步比较分析了这3种组织质膜蛋白的异同之处,并选取其中的5个蛋白质利用基质辅助激光解吸/电离飞行时间质谱仪成功进行了鉴定。一套完整的同时适用于茅苍术根、茎、叶各个组织的从高纯度质膜蛋白的制备到膜蛋白的质谱鉴定的蛋白质组学技术平台被成功构建。

In order to understand related metabolic pathways of secondary metabolites and carry on metabolic regulation of active medical ingredients in endangered medicinal plant Atractylodes lancea, the method of plasma membrane proteomics was studied in this paper. Purified plasma membrane proteins were respectively extracted and purified from root, stem, and leaf of A. lancea using uhracentrifugation combined with aqueous two-phase partitioning. Moreover, the conditions for two-dimensional electrophoresis （2DE） analysis were Optimized, and partial proteins in the latter were identified. The results showed that treating the ultra-centrifuged crude membrane compositions with the aqueous two-phase system of 6.4%, 6.3%, and 6.1% Dextran T-500/PEG 3350 （w/w） respectively, the high- purity plasma membrane proteins of 92.1% （root）, 91.5% （stem） and 90.8% （leaf） were obtained. The plasma membrane proteins were decomposed by 2% CHAPS and 2% Triton X-100 as the comprehensive detergents, loaded in the amount of 100 ~tg and resolved by IEF of 80 000 Vhs and 12.5%-15% SDS-PAGE gradient gel, and total 267, 297 and 248 protein spots in the plasma membrane protein profiles of root, stem and leave respectively were detected by the Image Master 2D Platinum 7.0 software. And similarities and differences of 3 tissue proteins were further comparatively analyzed. At last, 5 proteins were selected and successfully identified by MALDI-TOF-MS. Conclusively, a complete proteomics technology platform for preparing high-purity plasma membrane proteins from varied tissues of root, stem and leave ofA. lancea and identifying the mass spectrometry of membrane protein is established.