Zhao Yingxin, Zhang Wei, Kho Yoonjung, Zhao Yingming
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9038, USA.
Anal Chem. 2004 Apr 1;76(7):1817-23. doi: 10.1021/ac0354037.
Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.
对于鉴定疾病细胞中过表达的蛋白质而言，亟需高效的质膜整合蛋白分析方法。此类方法将有助于理解基本生物学过程，并发现用于治疗性单克隆抗体设计的蛋白质靶点。避免亚细胞器和胞质蛋白的污染对于成功进行质膜整合蛋白的蛋白质组学分析至关重要。在此，我们报告一种用于制备质膜整合蛋白的生物素导向亲和纯化（BDAP）方法，该方法包括：（1）对活细胞表面膜蛋白进行生物素化；（2）使用链霉亲和素磁珠进行亲和富集；（3）用高盐和高pH缓冲液进行严苛洗涤以去除与质膜相关的胞质蛋白。然后提取质膜整合蛋白，进行SDS-PAGE分离和HPLC/MS/MS分析以鉴定蛋白质。我们使用BDAP方法从人肺癌细胞系制备质膜整合蛋白。蛋白质印迹分析表明，该制备物几乎完全不含肌动蛋白（一种主要的胞质蛋白）。对从亲和富集的质膜整合蛋白制备物中提取的仅30微克蛋白质进行纳米HPLC/MS/MS分析，鉴定出898种独特蛋白质，其中781种根据其质膜定位进行了注释。在注释的蛋白质中，至少526种（67.3%）是质膜整合蛋白。其中值得注意的是62种异戊二烯化蛋白和45种Ras家族蛋白。据我们所知，这是迄今为止对哺乳动物细胞质膜整合蛋白最全面的蛋白质组学分析。鉴于整合膜蛋白在药物设计中的重要性，所述方法将加快质膜亚蛋白质组的表征以及质膜蛋白药物靶点的发现。
