Department of Chemistry, Fudan University, Shanghai, P R China.
Proteomics. 2011 Sep;11(17):3482-90. doi: 10.1002/pmic.201000211. Epub 2011 Jul 21.
This study developed a simple and rapid purification method for plasma membrane with high yields from adherent cells. The plasma membrane (PM) sheets could be absorbed specifically by the cationic silica-magnetite nanocomposites (CSMN) under acidic conditions, and recovered directly in cell-lysis-buffer with no need for precipitation. The binding between CSMN and PM sheets was confirmed by electron microscopy. Western blot analysis demonstrated a >10-fold relative enrichment factor. Up to 422 integral membrane proteins were identified from 10(7) Huh7 cells. Notably, we found 29 Ras family proteins by classification according to their biological functions. The whole enrichment procedure took <30 min. The CSMN-based procedure demonstrates a simple, economical and efficient enrichment of integral PM proteins in proteomic study.
本研究开发了一种从贴壁细胞中高效提取质膜的简单、快速的方法。在酸性条件下，质膜（PM）片可被带正电荷的硅磁铁矿纳米复合材料（CSMN）特异性吸附，并直接回收于裂解缓冲液中，无需沉淀。CSMN 与 PM 片之间的结合通过电子显微镜得到证实。Western blot 分析表明，相对富集因子大于 10 倍。从 10(7)个 Huh7 细胞中鉴定出多达 422 种完整的膜蛋白。值得注意的是，我们根据其生物学功能对 29 种 Ras 家族蛋白进行了分类。整个富集过程耗时<30 分钟。基于 CSMN 的方法在蛋白质组学研究中展示了一种简单、经济、高效的整体质膜蛋白富集方法。
