Molecular Bioenergetics Group, Medical School, Cluster of Excellence Frankfurt Macromolecular Complexes, Centre for Membrane Proteomics, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Proteomics. 2010 Apr;10(7):1401-7. doi: 10.1002/pmic.200900756.
We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN-PAGE) and laser-induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent-tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 microg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small-scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.
我们开发了一种实验方法，将两种强大的方法结合用于大膜蛋白复合物的蛋白质组学分析：蓝色 native 电泳 (BNE 或 BN-PAGE) 和激光诱导液珠离子解吸 (LILBID) MS。通过 BNE 分离蛋白质复合物并从凝胶中洗脱。通过 LILBID MS 获得多蛋白复合物的组成部分的质量，这是一种耐受去污剂的方法，特别适合于膜蛋白的表征。LILBID MS 需要高灵敏度和小体积的样品，因此对样品量的要求较低。从单个条带的洗脱液可以评估整个多蛋白复合物及其亚基的质量。该方法已通过来自 Yarrowia lipolytica 的线粒体 NADH：泛醌还原酶进行了验证。对于这个 947 kDa 的复合物，通常需要 30 微克或 32 皮摩尔即可获得可以分析亚基组成的光谱。通过在 BNE 的第二维上进一步分离，这种电泳小规模纯化天然复合物的方法的分辨率得到了显著提高。从通过差速离心获得的亚细胞级分开始，这允许在单个 LILBID 光谱中纯化和分析大的多蛋白复合物的组成部分。
