Affinity enrichment of plasma membrane for proteomics analysis
Affinity enrichment of plasma membrane for proteomics analysis
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DOI：
10.1002/elps.200305569
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发表时间：
2003-08-01
期刊：
影响因子：
2.9
通讯作者：
Zhao, YM
中科院分区：
文献类型：
--
作者：
Zhang, W;Zhou, G;Zhao, YM
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，可能是鉴定对这些环境作出反应的膜相关蛋白的有力方法。制备低细胞器污染的高浓度质膜组分是这类研究的必要条件。在这里，我们描述了一种亲和富集方法，该方法结合了细胞表面生物素化和固定化链霉亲和素珠的亲和富集，用于分离质膜。该方法使质膜相对于内质网富集400倍，内质网是标准质膜制剂中的主要污染物，并且显著减少了来自其他细胞器的污染。生物素化反应不会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，这表明细胞表面信号转导机制仍然有效。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良好的起始材料，如信号分子的动态运输和调节研究或疾病特异性膜标记物的鉴定。
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.