Schindler Jens, Lewandrowski Urs, Sickmann Albert, Friauf Eckhard, Nothwang Hans Gerd
Animal Physiology Group, Department of Biology, University of Kaiserslautern, 67653 Kaiserslautern, Germany.
Mol Cell Proteomics. 2006 Feb;5(2):390-400. doi: 10.1074/mcp.T500017-MCP200. Epub 2005 Oct 24.
A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases. Proteomics offers the potential to perform such a comprehensive analysis, yet it requires efficient protocols for the purification of the plasma membrane compartment. Here, we present a novel and efficient protocol for the separation and enrichment of brain plasma membrane proteins. It lasts only 4 h and is easy to perform. It highly enriches plasma membrane proteins and can be applied to small amounts of brain tissue, such as the cerebellum of a single rat, which was used in the present study. The protocol is based on affinity partitioning of microsomes in an aqueous two-phase system. Marker enzyme assays demonstrated a more than 12-fold enrichment of plasma membranes and a strong reduction of other compartments, such as mitochondria and the endoplasmic reticulum. 506 different proteins were identified when the enriched proteins underwent LC-MS/MS analysis subsequent to protein separation by SDS-PAGE. Using gene ontology, 146 proteins were assigned to a subcellular compartment. Ninety-three of those (64%) were membrane proteins, and 49 (34%) were plasma membrane proteins. A combined literature and database search for all 506 identified proteins revealed subcellular information on 472 proteins, of which 197 (42%) were plasma membrane proteins. These comprised numerous transporters, channels, and neurotransmitter receptors, e.g. the inward rectifying potassium channel Kir7.1 and the cerebellum-specific gamma-aminobutyric acid receptor GABRA6. Surface proteins involved in cell-cell contact and disease-related proteins were also identified. Six of the 146 assigned proteins were derived from mitochondrial membranes and 5 from membranes of the endoplasmic reticulum. Taken together, our protocol represents a simple, rapid, and reproducible tool for the proteomic characterization of brain plasma membranes. Because it conserves membrane structure and protein interactions, it is also suitable to enrich multimeric protein complexes from the plasma membrane for subsequent analysis.
对质膜蛋白进行全面分析对于深入了解大脑发育、功能及疾病至关重要。蛋白质组学为开展此类全面分析提供了可能，但需要高效的质膜区室纯化方案。在此，我们提出一种新颖且高效的大脑质膜蛋白分离与富集方案。该方案仅需4小时，操作简便。它能高度富集质膜蛋白，可应用于少量脑组织，如本研究中使用的单只大鼠的小脑。该方案基于微粒体在水相双相系统中的亲和分配。标记酶分析表明，质膜富集了超过12倍，而线粒体和内质网等其他区室则大幅减少。通过SDS-PAGE分离蛋白质后，对富集的蛋白质进行LC-MS/MS分析，鉴定出506种不同的蛋白质。利用基因本体论，将146种蛋白质归入亚细胞区室。其中93种（64%）为膜蛋白，49种（34%）为质膜蛋白。对所有506种已鉴定蛋白质进行文献和数据库综合检索，揭示了472种蛋白质的亚细胞信息，其中197种（42%）为质膜蛋白。这些蛋白质包括众多转运体、通道和神经递质受体，例如内向整流钾通道Kir7.1和小脑特异性γ-氨基丁酸受体GABRA6。还鉴定出了参与细胞间接触的表面蛋白和疾病相关蛋白。在这146种归入的蛋白质中，有6种源自线粒体膜，5种源自内质网膜。综上所述，我们的方案是一种用于大脑质膜蛋白质组学表征的简单、快速且可重复的工具。由于它保留了膜结构和蛋白质相互作用，也适合从质膜中富集多聚体蛋白复合物以供后续分析。
