title: A straightforward and efficient analytical pipeline for metaproteome characterization.
abstract: 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 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.
  
  
  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.
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tanca
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Palomba
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Pisanu
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Deligios
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Fraumene
authors.affiliation: Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Manghina
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Pagnozzi
authors.affiliation: Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio 07041 Alghero, Italy.
authors.forename: Maria Filippa
authors.initials: MF
authors.lastname: Addis
authors.affiliation: 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.
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
keywords: Gut microbiota; LC-MS/MS; Mass spectrometry; Metaproteomics; Microbial community; Mouse; Protein extraction; Proteomic methods; Shotgun proteomics; Single-run liquid chromatography