Herald V L, Heazlewood J L, Day D A, Millar A H
Plant Molecular Biology Group, School of Biomedical and Chemical Sciences, The University of Western Australia, Crawley, WA 6009, Australia.
FEBS Lett. 2003 Feb 27;537(1-3):96-100. doi: 10.1016/s0014-5793(03)00101-7.
Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of divalent cations during two-dimensional diagonal sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Tandem mass spectrometry and searches of the predicted Arabidopsis protein dataset were used in an attempt to identify 34 of the proteins which shifted. This analysis identified a total of 23 distinct protein spots as the products of at least 11 different Arabidopsis genes. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identified. These included: succinyl CoA ligase beta subunit, Mn-superoxide dismutase (SOD), an Fe-S centred component of complex I and the REISKE iron-sulphur protein of the b/c(1) complex. A further set of four proteins of known function but without known divalent binding properties were also identified: the Vb subunit of cytochrome c oxidase, a subunit of ATP synthase (orfB), the acyl carrier protein, and the translocase of the outer membrane (TOM20). Three other proteins, of unknown function, were also found to shift in the presence of divalent cations. This approach has broad application for the identification of sub-proteomes based on the metal interaction of polypeptides.
在二维对角线十二烷基硫酸钠-聚丙烯酰胺凝胶电泳过程中，通过在二价阳离子存在下的迁移率变化，对拟南芥线粒体蛋白质组中的二价金属结合蛋白进行了分析。串联质谱分析以及对拟南芥预测蛋白质数据集的搜索，旨在鉴定34种发生迁移的蛋白质。该分析共鉴定出23个不同的蛋白质斑点，它们是至少11个不同拟南芥基因的产物。鉴定出了一系列已知为二价阳离子结合蛋白或催化二价阳离子依赖性反应的蛋白质。这些包括：琥珀酰辅酶A连接酶β亚基、锰超氧化物歧化酶（SOD）、复合物I的一个以铁硫为中心的组分以及b/c(1)复合物的 Rieske 铁硫蛋白。还鉴定出另外一组四种功能已知但无二价结合特性的蛋白质：细胞色素c氧化酶的Vb亚基、ATP合酶的一个亚基（orfB）、酰基载体蛋白和外膜转位酶（TOM20）。另外还发现三种功能未知的蛋白质在二价阳离子存在下也会发生迁移。这种方法对于基于多肽的金属相互作用鉴定亚蛋白质组具有广泛的应用。
