Relating human gut metagenome and metaproteome
Gut microbiome research is rapidly moving towards the functional characterization of the microbiota by means of shotgun meta-omics. Here, we selected a cohort of healthy subjects from an indigenous and monitored Sardinian population to analyze their gut microbiota using both shotgun metagenomics and shotgun metaproteomics. We found a considerable divergence between genetic potential and functional activity of the human healthy gut microbiota, in spite of a quite comparable taxonomic structure revealed by the two approaches. Investigation of inter-individual variability of taxonomic features revealed Bacteroides and Akkermansia as remarkably conserved and variable in abundance within the population, respectively. Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the functional activity with the higher expression rate and the lower inter-individual variability in the study cohort, highlighting the key importance of the biosynthesis of this microbial by-product for the gut homeostasis. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several gut microbiota members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis and short-chain fatty acid production). In conclusion, our results provide useful indications regarding the main functions actively exerted by the gut microbiota members of a healthy human cohort, and support metaproteomics as a valuable approach to investigate the functional role of the gut microbiota in health and disease.
Sample Processing Protocol
Samples. Stool samples were collected from 15 healthy Sardinian volunteers selected to avoid age, sex and BMI biases (SI Table S1) and immediately stored at -80°C. Two equal portions were collected from each sample and subjected to DNA and protein extraction, respectively. Metaproteome analysis. Proteins were extracted (1) and digested (2) as described previously. LC-MS/MS analysis was carried out using an LTQ-Orbitrap Velos mass spectrometer interfaced with an UltiMate 3000 RSLCnano LC system (both from Thermo Scientific). (1) Tanca A, et al. (2014) A straightforward and efficient analytical pipeline for metaproteome characterization. Microbiome 2(1):49. (2) Tanca A, Biosa G, Pagnozzi D, Addis MF, & Uzzau S (2013) Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome. Proteomics 13(17):2597-2607.
Data Processing Protocol
Peptide identification was performed using Sequest-HT and Percolator, embedded in Proteome Discoverer™, as described previously (3). (3) Tanca A, Palomba A, Pisanu S, Addis MF, & Uzzau S (2015) Enrichment or depletion? The impact of stool pretreatment on metaproteomic characterization of the human gut microbiota. Proteomics 15(20):3474-3485.
Corresponding dataset(s) in other omics resources
PRJEB19090 (ENA, EMBL-EBI)
Tanca A, Abbondio M, Palomba A, Fraumene C, Manghina V, Cucca F, Fiorillo E, Uzzau S. Potential and active functions in the gut microbiota of a healthy human cohort. Microbiome. 2017 Jul 14;5(1):79 PubMed: 28709472