Metaproteome of the waste water treatment plant Gerwisch/Germany

In this study the impact of protein fractionation techniques prior to LC/MS analysis was investigated on activated sludge samples derived at winter and summer condition from a full-scale wastewater treatment plant (WWTP). For reduction of the sample complexity, different fractionation techniques including RP-LC (1D-approach), SDS-PAGE and RP-LC (2D-approach) as well as RP-LC, SDS-PAGE and liquid IEF (3D-approach) were carried out before subsequent ITMS analysis. The derived spectra were identified by MASCOT search using a combination of the public UniProtKB/Swiss-Prot protein database and metagenome data from a WWTP (data are available via ProteomeXchange with identifier PXD001547). The results showed a significant increase of identified spectra, enabled by applying IEF and SDS-PAGE to the proteomic workflow. Based on meta-proteins, a core metaproteome and the corresponding taxonomic profile of the wastewater activated sludge was revealed and functional aspects using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway library were analyzed. Hereby, the KEGG Orthology identifiers (KO numbers) of protein hits were plotted into pathway maps of the central carbon (map01200) and nitrogen metabolism (map00910). Using the 3D-approach, most proteins involved in glycolysis and citrate cycle and nearly all proteins of the nitrogen removal were identified, qualifying this approach as the most promising for future studies.

本研究针对某全规模运行的大型污水处理厂（Wastewater Treatment Plant, WWTP）在冬季与夏季运行工况下采集的活性污泥样本，探究了液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LC/MS）分析前采用的蛋白质分级分离技术的影响效果。为降低样本复杂度，在后续离子阱质谱（Ion Trap Mass Spectrometry, ITMS）分析前，本研究采用了多种分级分离方案，包括反相液相色谱（Reversed-Phase Liquid Chromatography, RP-LC，一维方案）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE）结合反相液相色谱（二维方案），以及反相液相色谱、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结合液相等电聚焦（Isoelectric Focusing, IEF，三维方案）。所获得的质谱谱图通过MASCOT搜索引擎进行鉴定，检索库结合了公共UniProtKB/Swiss-Prot蛋白质数据库与该污水处理厂的宏基因组数据（相关数据可通过ProteomeXchange数据库获取，标识符为PXD001547）。研究结果显示，在蛋白质组学流程中引入等电聚焦与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳可显著提升可鉴定质谱谱图的数量。基于宏蛋白质数据，本研究解析了污水活性污泥的核心宏蛋白质组及其对应分类学特征，并借助京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路库对其功能特性展开分析。在此基础上，将鉴定到的蛋白质对应的KEGG直系同源簇标识符（KO编号）映射到中心碳代谢（map01200）与氮代谢（map00910）通路图谱中。采用三维分级分离方案时，糖酵解与三羧酸循环相关的绝大多数蛋白质，以及几乎全部脱氮相关蛋白质均可被鉴定，表明该方案为未来相关研究中极具应用前景的技术路径。