Fandiño Anabel S, Rais Isam, Vollmer Martin, Elgass Helmuth, Schägger Hermann, Karas Michael
Agilent Technologies R and D and Marketing GmbH and Co. KG, 76337 Waldbronn, Germany.
J Mass Spectrom. 2005 Sep;40(9):1223-31. doi: 10.1002/jms.903.
The application of two-dimensional electrophoresis for the identification of hydrophobic membrane proteins is principally hampered by precipitation of many of these proteins during first-dimension, isoelectric focusing. Therefore new strategies towards the identification and characterization of membrane proteins are being developed. In this work we present a direct and rapid approach from blue-native gels to mass spectrometry, which allows the analyses of complete complexes and prevents protein aggregation of hydrophobic regions during electrophoresis. We combine blue-native gel electrophoresis and liquid chromatography--nanospray-iontrap tandem mass spectrometry to analyze the composition of oxidative phosphorylation complexes I, III, IV and V from bovine-heart mitochondria as a model system containing a number of highly hydrophobic proteins. Bands from blue-native gels were subjected either to in-gel or to in-solution tryptic digestion. The obtained peptide mixtures were further analyzed by liquid chromatography--tandem mass spectrometry and the corresponding proteins were identified by database search. From a total of 86 proteins, 67 protein subunits could be identified including all highly hydrophobic components, except the ND4L and ND6 subunits of complex I. We demonstrate that liquid chromatography--tandem mass spectrometry combined to blue-native electrophoresis is a straightforward tool for proteomic analysis of multiprotein complexes, and especially for the identification of very hydrophobic membrane protein constituents that are not accessible by common isoelectric focusing/sodium dodecyl sulphate gel electrophoresis.
二维电泳用于鉴定疏水膜蛋白时，主要受到许多此类蛋白在第一维等电聚焦过程中沉淀的阻碍。因此，正在开发鉴定和表征膜蛋白的新策略。在这项工作中，我们提出了一种从蓝色天然凝胶到质谱的直接快速方法，该方法能够分析完整复合物，并防止电泳过程中疏水区域的蛋白聚集。我们将蓝色天然凝胶电泳与液相色谱-纳喷离子阱串联质谱相结合，以牛心线粒体中的氧化磷酸化复合物I、III、IV和V为模型系统进行分析，该模型系统包含许多高度疏水的蛋白。蓝色天然凝胶中的条带进行胶内或溶液内胰蛋白酶消化。所得肽混合物通过液相色谱-串联质谱进一步分析，并通过数据库搜索鉴定相应蛋白。在总共86种蛋白中，可鉴定出67种蛋白亚基，包括所有高度疏水成分，但复合物I的ND4L和ND6亚基除外。我们证明，液相色谱-串联质谱与蓝色天然电泳相结合是蛋白质组学分析多蛋白复合物的一种直接工具，尤其适用于鉴定普通等电聚焦/十二烷基硫酸钠凝胶电泳无法检测到的高度疏水膜蛋白成分。
