厌氧产氢颗粒污泥蛋白质组分析样品的高效制备方法

Efficient preparation method of samples for proteomic analysis of anaerobic hydrogen-producing granular sludge

【背景】厌氧产氢颗粒污泥比絮状产氢污泥具有更高的生物量、沉降性与反应效率,对颗粒污泥进行蛋白质组学研究,有助于揭示其代谢调控的分子机制,从而对厌氧代谢过程进行优化调控。目前关于产氢颗粒污泥蛋白质组分析样品制备方法的研究尚未见文献报道。革兰氏阳性菌Ethanoligenens harbinense YUAN-3是自凝集产氢发酵细菌,在间歇和连续流培养中可形成自聚集的厌氧颗粒,由于其全基因组信息清楚,可作为模式研究材料对制备方法进行评估。【目的】针对厌氧产氢颗粒污泥的蛋白质组学研究,比较不同蛋白质提取方法进行优化。【方法】分别利用液氮研磨、超声破碎、匀浆破碎对产氢颗粒污泥破碎,比较这3种方法对总蛋白提取量的影响;通过双向电泳比较三氯乙酸(Trichloroacetic acid,TCA)-丙酮沉淀法与苯酚抽提法对总蛋白提取效果的影响;对总蛋白样品分别进行同位素标记相对和绝对定量标记(Isobaric tags for relative and absolute quantification,iTRAQ)、串联质谱标签(Tandem mass tag,TMT)标记以及质谱鉴定。【结果】液氮研磨、超声破碎、匀浆破碎3种破碎方法下总蛋白的提取量分别是对照样品的2.0、3.9与5.2倍。与TCA-丙酮沉淀法相比,苯酚抽提法总蛋白样品在双向电泳图谱上的蛋白质点明显增多,分布均匀,同时其在碱性蛋白端与小分子量蛋白端的蛋白质点也明显增多。质谱分析发现,iTRAQ标记样品与TMT标记样品中分别鉴定到1797个与1644个蛋白,在分子量、等电点、亚细胞定位的各个分布范围内,这些蛋白良好地覆盖了E.harbinense YUAN-3中各个类型的蛋白。【结论】匀浆破碎与苯酚抽提法联用的总蛋白制备方法更适用于厌氧产氢颗粒污泥,该方法有利于后续的蛋白质双向电泳和定量蛋白质组质谱分析,可作为产氢颗粒污泥以及革兰氏阳性菌总蛋白制备的方法参考。

[Background] Anaerobic hydrogen-producing granular sludge has more biomass, better settleability and hydrogen production efficiency than flocculent sludge. Proteomic study on hydrogen-producing granular sludge is helpful to reveal the molecular mechanism of microbial metabolic changes in granular sludge,which is beneficial to optimize the anaerobic metabolism process. However, sample preparation method for its proteomic analysis has not been reported. The gram positive bacterium Ethanoligenens harbinense YUAN-3 has been the only reported auto-aggregative hydrogen-producing strain, which can form hydrogen-producing granular in batch and continuous cultures. The genome of strain YUAN-3 has been completely sequenced. Therefore, granules which were formed by strain YUAN-3 were used as model samples in the following experiments.[Objective] In order to facilitate proteomic research on hydrogen-producing granular sludge, protein sample preparation method for proteomic analysis was optimized.[Methods] Protein yields from liquid nitrogen grinding, sonication, and homogenization on was investigated. Protein quality of samples prepared by trichloroacetic acid(TCA)-acetone precipitation method and phenol extraction method was determined by Two-dimensional gel electrophoresis. The proteins were further labeled with iTRAQ(Isobaric tags for relative and absolute quantification) or TMT(Tandem mass tag) reagent and analyzed by using mass spectrometry.[Results] The protein yield from liquid nitrogen grinding, sonication, and homogenization were 2-fold, 3.9-fold, and 5.2-fold higher than the control respectively. Compared with TCA-acetone precipitation method, the protein spots, especially including basic proteins and low molecular weight proteins, significantly increased and uniformly distributed in the phenol extraction sample. 1 797 and 1 644 proteins were identified in the iTRAQ and TMT labeling samples respectively compared to 2 701 predicted proteins from E. harbinense YUAN-3. The distribution of identified proteins across different ranges of molecular weight, isoelectric point and subcellular localization was similar to the predicted proteome of E. harbinense YUAN-3 based on its genome with some exceptions.[Conclusion] Combination of homogenization and phenol extraction method works the best in sample preparation for proteomic analysis of hydrogen-producing granular sludge, which contributes to obtain high quality proteomic data.