Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase.
Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase.
复制标题
DOI：
10.1002/jms.467
复制
发表时间：
2003-05
期刊：
影响因子：
--
通讯作者：
G. Sun;M. Kinter;V. Anderson
中科院分区：
文献类型：
--
作者：
G. Sun;M. Kinter;V. Anderson
复合物III的线粒体电子传递链，泛喹啉-细胞色素c还原酶，分离出蓝色的天然聚丙烯酰胺凝胶电泳。10的11个多肽存在于这个复杂的直接检测基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）电洗脱后的活性复合物。胰蛋白酶和胰凝乳蛋白酶消化的复合物允许从所有的蛋白质亚基与70%的覆盖率的250 kDa的复合物的特定肽的识别。所有11种蛋白质的质量通过二维Tricine十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和分离的多肽的洗脱来确认。此外，通过SDS-PAGE分离蛋白质的凝胶内胰蛋白酶消化产生的肽的液相色谱/串联质谱（LC/MS/MS）表征，确认了核心I、核心II、细胞色素c和Rieske铁硫蛋白的同一性。这种膜结合的电子传递复合物的分析方法应适用于其他膜复合物，特别是其他线粒体电子传递复合物。通过凝胶内消化完整复合物获得的肽的MS分析允许同时表征天然蛋白质和修饰，所述修饰导致线粒体缺陷，所述线粒体缺陷被认为是导致病理状况的原因。
Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.