A straightforward and efficient analytical pipeline for metaproteome characterization

Background: 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.Results: 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 10⁴ to 10⁸ 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.Conclusions: 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.