Functional metaproteome analysis of protein extracts from contaminated soil and groundwater
Functional metaproteome analysis of protein extracts from contaminated soil and groundwater
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DOI：
10.1038/ismej.2007.39
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发表时间：
2007-07-01
期刊：
影响因子：
11
通讯作者：
von Bergen, Martin
中科院分区：
文献类型：
--
作者：
Benndorf, Dirk;Balcke, Gerd U.;von Bergen, Martin
利用土壤或地下水中的蛋白质作为功能性生物标志物需要高效的提取。我们开发了一种提取方法，该方法通过0.1M NaOH处理和苯酚萃取相结合，实现了土壤基质中无机和有机成分的蛋白质分离。与NaOH孵育释放腐植酸和蛋白质从土壤矿物质，同时，破坏微生物。随后的苯酚萃取将蛋白质从腐殖质有机物中分离出来。蛋白提取物分别进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS- PAGE）和二维电泳（2- DE）。通过电喷雾电离源（LC- ESI- MS）在线质谱联用液相色谱（LC- ESI- MS）对斑点和条带进行分离，鉴定单个蛋白。为了评估该方法对环境超蛋白质组功能分析的适用性，将其应用于富含氯苯氧基酸降解细菌的土壤中，并与2,4-二氯苯氧基乙酸（2,4- D）孵育22天。该方法还应用于某氯苯污染场地含水层的地下水分析。氯儿茶酚双加氧酶等酶的鉴定与这些样品中预期表达的细菌代谢途径一致。因此，该方案使土壤和地下水样品的元蛋白质组分析成为可能。因此，它提供了一种研究环境微生物群落多样性的方法，同时比元基因组甚至元转录组分析更直接地解决功能方面的问题。
Using proteins from soil or groundwater as functional biomarkers requires efficient extraction. We developed an extraction method in which the separation of proteins from the inorganic and organic constituents of the soil matrix was achieved by a combination of 0.1M NaOH treatment and phenol extraction. Incubation with NaOH released humic acids and proteins from soil minerals, and simultaneously, disrupted microorganisms. The subsequent phenol extraction separated the proteins from the humic organic matter. Protein extracts were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS- PAGE) and 2D- electrophoresis ( 2- DE). Spots and bands were excised and individual proteins identified by liquid chromatography online linked to mass spectrometry ( MS) via electrospray ionization source ( LC- ESI- MS). To assess the suitability of the method for the functional analysis of environmental metaproteomes, it was applied to soil that had been enriched in chlorophenoxy acid- degrading bacteria by incubation with 2,4- dichlorophenoxy acetic acid ( 2,4- D) for 22 days. The method was also used to analyze groundwater from the aquifer of a chlorobenzene- contaminated site. The identification of enzymes such as chlorocatechol dioxygenases was consistent with bacterial metabolic pathways expected to be expressed in these samples. The protocol enabled thus the analysis of the metaproteome of soil and groundwater samples. It thereby provides a means to study the diversity of environmental microbial communities while addressing functional aspects more directly than metagenome or even metatranscriptome analysis.