Proteomic analysis of integral plasma membrane proteins
Proteomic analysis of integral plasma membrane proteins
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DOI：
10.1021/ac0354037
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发表时间：
2004-04-01
影响因子：
7.4
通讯作者：
Zhao, YM
中科院分区：
文献类型：
--
作者：
Zhao, YX;Zhang, W;Zhao, YM
对于鉴定疾病细胞中过度表达的蛋白质，非常需要有效的方法来分析质膜上的蛋白质。这些方法将有助于理解基本的生物学过程和发现治疗性单克隆抗体设计的蛋白质靶点。避免被亚细胞器和细胞质蛋白污染是成功分析完整质膜蛋白的关键。在这里，我们报道了一种生物素导向的亲和纯化（BDAP）方法，用于制备完整的质膜蛋白，其中包括(1)活细胞表面膜蛋白的生物素化，(2)使用链霉亲和素珠进行亲和富集，以及(3)用高盐和高ph缓冲液进行强烈洗涤，去除质膜相关的胞质蛋白。提取完整的质膜蛋白，进行SDS-PAGE分离和HPLC/MS/MS鉴定。我们用BDAP法制备了人肺癌细胞系的完整质膜蛋白。Western blotting分析表明，该制剂几乎完全不含肌动蛋白，这是一种主要的细胞质蛋白。纳米高效液相色谱/质谱/质谱分析仅从亲和力富集的整体质膜制备中提取的30杯蛋白质，鉴定出898个独特的蛋白质，其中781个蛋白质在质膜上进行了定位注释。其中至少526个（67.3%）为完整质膜蛋白。其中，有62种戊酰化蛋白和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中整体质膜蛋白进行的最全面的蛋白质组学分析。鉴于整体膜蛋白对药物设计的重要性，所描述的方法将加速质膜亚蛋白质组的表征和发现质膜蛋白药物靶点。
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 mug 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.