Comparative membrane metalloproteomics of respiring anaerobes

厌氧呼吸微生物的能量守恒高度依赖于含金属的多聚体膜结合蛋白复合物，它允许电子供体的氧化、电子的结构化传导和末端电子受体的还原。虽然许多呼吸复合物已在选定的生物体中进行了详细的研究，但金属蛋白、含金属辅助因子和金属离子在呼吸厌氧菌中的总体分布尚不清楚。然而，在厌氧菌基因组中编码的大量金属蛋白复合物，预计位于膜中，没有相关功能。含mo的FeS簇蛋白和11亚基复合物I同源物都在原核生物基因组中大量编码。我们提出利用电感耦合等离子体质谱法（ICP-MS）和液相色谱质谱法（LC-MS/MS）分别定量金属和蛋白质亚基，研究膜结合金属蛋白复合物在多种原核生物中的存在和分布。本研究将包括三个工作包：第一，全细胞膜制备中金属和蛋白质的比较分析，即散弹枪膜金属蛋白质组学；二是天然凝胶电泳分离金属蛋白配合物后，对金属蛋白配合物中的金属和蛋白质进行对比分析；第三，与金属蛋白相关的含金属有机辅因子的质谱分析。我们将重点研究含Fe， Ni， Co和Mo金属的蛋白质复合物以及含金属的辅助因子F430，钴胺素和钼翼素，它们都参与厌氧节能过程的关键反应。该项目与SPP内提出的一系列其他项目在主题和方法上都有联系。通过对细菌的研究，我们可以将这些组的结果整合到我们的工作中，以构建金属蛋白质组学的整体图景，同时为这些组提供补充信息。其他合作课题包括Mo-cofactor和F430 cofactor分析，以及大肠杆菌中金属蛋白的异源过表达和组装检测。最后，展望了在金属质谱、蛋白质质谱和次级离子质谱（nanoSIMS, ToF-SIMS）方面的方法学合作。

Energy conservation in anaerobically respiring microorganisms highly depends on metal-containing multimeric membrane-bound protein complexes allowing the oxidation of electron donors, the structured conduction of electrons and the reduction of terminal electron acceptors. Although many respiratory complexes have been studied in detail in selected organisms, the overall distribution of metalloproteins, metal-containing cofactors and metal ions in respiring anaerobes is unknown. Still, a significant number of metalloprotein complexes encoded in the genomes of anaerobes and predicted to be located in the membrane have no affiliated function. The Mo-containing FeS cluster proteins and the 11-subunit complex I homologues, both abundantly encoded in prokaryotic genomes, are among them. We propose to study the occurrence and distribution of membrane-bound metalloprotein complexes in diverse prokaryotes by ICP-MS (inductively coupled plasma mass spectrometry) and LC-MS/MS (liquid chromatography mass spectrometry) to quantify metals and protein subunits, respectively. The study will consist of three work packages: First, the comparative analysis of metals and proteins in membrane preparations of whole cells, i.e. shotgun membrane metalloproteomics; second, the comparative analysis of metals and proteins in metalloprotein complexes after separation of the complexes by native gel electrophoresis; and third, the mass spectrometric analysis of metal-containing organic cofactors associated with metalloproteins. We will focus on protein complexes containing the metals Fe, Ni, Co and Mo and the metal-containing cofactors F430, cobalamin and Mo-pterins, all involved in key reactions of anaerobic energy-conserving processes.The project is connected to a series of other projects proposed within the SPP, both with regard to their topic and methodologically. By investigating bacteria, in which specific metalloproteins are investigated by other groups of the SPP, we can integrate results of these groups into our work to assemble an overall picture of metalloproteomics and at the same time provide supplementary information to these groups. Other cooperative topics are Mo-cofactor and cofactor F430 analysis as well as the detection of heterologously overexpressed and assembled metalloproteins in E. coli. Finally, methodological cooperation in metal mass spectrometry, protein mass spectrometry and secondary ion mass spectrometry (nanoSIMS, ToF-SIMS) is projected.