Zhang Wei, Zhou Ge, Zhao Yingxin, White Michael A, Zhao Yingming
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA.
Electrophoresis. 2003 Aug;24(16):2855-63. doi: 10.1002/elps.200305569.
Proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially a powerful approach for the identification of membrane-associated proteins responding to these environments. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. Here, we describe an affinity enrichment method, which combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads, for the isolation of plasma membranes. This method results in a 400-fold enrichment of plasma membrane relative to endoplasmic reticulum, a major contaminant in standard plasma membrane preparations, and dramatically reduces contamination from other cellular organelles. The biotinylation reaction did not interfere with ligand-dependent activation of receptor tyrosine kinases or G-protein coupled receptors, suggesting cell-surface signal transduction machinery remains functional. Membrane fractions prepared by this method should provide excellent starting materials for membrane proteomics analysis such as studies of dynamic trafficking and regulation of signaling molecules or identification of disease-specific membrane markers.
对暴露于不同细胞外环境的细胞的质膜进行蛋白质组学分析，可能是鉴定响应这些环境的膜相关蛋白的一种强大方法。制备来自细胞器的低污染的高浓度质膜组分对于此类研究至关重要。在这里，我们描述了一种亲和富集方法，该方法将细胞表面生物素化与通过固定化链霉亲和素珠进行的亲和富集相结合，用于质膜的分离。相对于内质网（标准质膜制备中的主要污染物），该方法使质膜富集了400倍，并显著减少了来自其他细胞器的污染。生物素化反应不干扰受体酪氨酸激酶或G蛋白偶联受体的配体依赖性激活，表明细胞表面信号转导机制仍然具有功能。通过这种方法制备的膜组分应该为膜蛋白质组学分析提供极好的起始材料，例如研究信号分子的动态运输和调节或鉴定疾病特异性膜标记物。
