Two-dimensional Blue Native sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets
Two-dimensional Blue Native sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets
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DOI：
10.1002/elps.200406196
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发表时间：
2005-03-01
期刊：
影响因子：
2.9
通讯作者：
Meyer, BJ
中科院分区：
文献类型：
--
作者：
Claeys, D;Geering, K;Meyer, BJ
二维（2-D）蓝色天然/SDS凝胶电泳将天然状态下单体和多聚体蛋白质的第一维分离与第二维变性相结合。这些高分辨率2-D凝胶的目的是确定多蛋白复合物相对于其亚基组成。我们首次应用这种方法来分析两个人血小板亚蛋白质组：胞浆和微粒体膜蛋白组分。血小板膜蛋白的增溶是用去甲变性去污剂n-十二烷基-β-（D）-麦芽糖苷实现的。为了验证天然增溶条件，我们证明了正确的组装的Na，KATP酶，功能性多聚体跨膜蛋白，在非洲爪蟾卵母细胞中表达时。我们确定了63血小板蛋白后，在胶内胰蛋白酶消化的58个选定的蛋白点和液相色谱-串联质谱。9种蛋白质首次在血小板中通过蛋白质组学方法检测到。我们还表明，这种技术有效地解决了几个已知的膜和胞质多蛋白复合物。因此，蓝色天然/SDS凝胶电泳是一种有价值的程序，分析特定的血小板亚蛋白质组，如膜（结合）蛋白质部分，通过质谱和免疫印迹，并可能是相关的蛋白质-蛋白质相互作用的研究后血小板活化。
Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nordenaturing detergent n-dodecyl-beta-(D)-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,KATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.