Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy.
Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy.
Microbiome. 2014 Dec 10;2(1):49. doi: 10.1186/s40168-014-0049-2. eCollection 2014.
The massive characterization of host-associated and environmental microbial communities has represented a real breakthrough in the life sciences in the last years. In this context, metaproteomics specifically enables the transition from assessing the genomic potential to actually measuring the functional expression of a microbiome. However, significant research efforts are still required to develop analysis pipelines optimized for metaproteome characterization.
This work presents an efficient analytical pipeline for shotgun metaproteomic analysis, combining bead-beating/freeze-thawing for protein extraction, filter-aided sample preparation for cleanup and digestion, and single-run liquid chromatography-tandem mass spectrometry for peptide separation and identification. The overall procedure is more time-effective and less labor-intensive when compared to state-of-the-art metaproteomic techniques. The pipeline was first evaluated using mock microbial mixtures containing different types of bacteria and yeasts, enabling the identification of up to over 15,000 non-redundant peptide sequences per run with a linear dynamic range from 10(4) to 10(8) colony-forming units. The pipeline was then applied to the mouse fecal metaproteome, leading to the overall identification of over 13,000 non-redundant microbial peptides with a false discovery rate of <1%, belonging to over 600 different microbial species and 250 functionally relevant protein families. An extensive mapping of the main microbial metabolic pathways actively functioning in the gut microbiome was also achieved.
The analytical pipeline presented here may be successfully used for the in-depth and time-effective characterization of complex microbial communities, such as the gut microbiome, and represents a useful tool for the microbiome research community.
近年来，对宿主相关和环境微生物群落的大规模描述代表了生命科学的真正突破。在这种情况下，代谢蛋白质组学特别能够实现从评估基因组潜力到实际测量微生物组功能表达的转变。然而，仍然需要大量的研究努力来开发针对代谢蛋白质组学特性的分析管道。
这项工作提出了一种用于鸟枪法代谢蛋白质组学分析的有效分析管道，该管道结合了珠磨/冻融用于蛋白质提取、过滤辅助样品制备用于净化和消化、以及单运行液相色谱-串联质谱用于肽分离和鉴定。与最先进的代谢蛋白质组学技术相比，该整体程序更省时、劳动强度更低。该管道首先使用含有不同类型细菌和酵母的模拟微生物混合物进行评估，每个运行可鉴定多达 15000 个非冗余肽序列，线性动态范围从 10(4)到 10(8)个菌落形成单位。该管道随后应用于小鼠粪便代谢蛋白质组学，总共鉴定出超过 13000 个非冗余微生物肽，假发现率<1%，属于超过 600 种不同的微生物物种和 250 种功能相关的蛋白质家族。还实现了对肠道微生物组中活跃的主要微生物代谢途径的广泛映射。
这里提出的分析管道可成功用于复杂微生物群落（如肠道微生物组）的深入和高效特征描述，是微生物组研究界的有用工具。
