Reisinger Veronika, Eichacker Lutz Andreas
Ludwig Maximilian University, Munich, Germany.
Proteomics. 2007 Sep;7 Suppl 1:6-16. doi: 10.1002/pmic.200700205.
Natural compartmentalization makes proteome analysis of the cell, cell organelles and organelle subfractions possible. Protein complexes are the basis for the next level of compartmentalization that can be addressed well with proteomic technology. Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space. Cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism can be defined by the activity of protein complexes. Since a large part of the protein complexes of the cell body are inserted in lipid membrane phases, isolation, separation and protein subunit identification were difficult to address. Blue native polyacrylamide gel electrophoresis (BN-PAGE) provides us with the technology for high resolution separation of membrane protein complexes. Here, we show that high resolution separation of protein complexes by BN-PAGE requires the establishment of a detailed solubilisation strategy. We show that BN/SDS-PAGE provides the scientist with a high resolution array of protein subunits which allows analysis of the specific subunit stoichiometry of a protein complex as well as the assembly of protein complexes by standard protein detection methodology like DIGE, gelblot analysis and mass spectrometry. We envision BN-PAGE to precede classical 2D IEF/SDS-analysis for detailed characterization of membrane proteomes.
自然区室化使得对细胞、细胞器及细胞器亚组分进行蛋白质组分析成为可能。蛋白质复合物是下一级区室化的基础，蛋白质组学技术能够很好地解决这一问题。蛋白质复合物在时间和空间上的所有复杂层面组织并维持细胞和细胞器的功能。细胞发育与分裂、转录与翻译、呼吸与光合作用、运输与代谢都可以由蛋白质复合物的活性来定义。由于细胞体的大部分蛋白质复合物都嵌入脂质膜相中，因此其分离、提纯及蛋白质亚基鉴定一直难以解决。蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）为我们提供了高分辨率分离膜蛋白复合物的技术。在此，我们表明，通过BN-PAGE对蛋白质复合物进行高分辨率分离需要建立详细的增溶策略。我们表明，BN/SDS-PAGE为科学家提供了一系列高分辨率的蛋白质亚基，从而能够通过DIGE、凝胶印迹分析和质谱等标准蛋白质检测方法来分析蛋白质复合物的特定亚基化学计量以及蛋白质复合物的组装情况。我们设想在对膜蛋白质组进行详细表征时，BN-PAGE应先于经典的二维IEF/SDS分析。
