Tanca, Alessandro and Palomba, Antonio and Pisanu, Salvatore and Deligios, Massimo and Fraumene, Cristina and Manghina, Valeria and Pagnozzi, Daniela and Addis, Maria Filippa and Uzzau, Sergio (2014) A Straightforward and efficient analytical pipeline for metaproteome characterization. Microbiome, Vol. 2 (1), p. 49. eISSN 2049-2618. Article.
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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
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.
|Uncontrolled Keywords:||Gut microbiota, microbial community, mouse, metaproteomics, protein extraction, proteomic methods, shotgun proteomics, mass spectrometry, LC-MS/MS, single-run liquid chromatography|
|Subjects:||Area 06 - Scienze mediche > MED/07 Microbiologia e microbiologia clinica|
|Divisions:||002 Altri enti e centri di ricerca del Nord Sardegna > Porto Conte Ricerche, Alghero|
001 Università di Sassari > 01-a Nuovi Dipartimenti dal 2012 > Scienze Biomediche
|Copyright Holders:||© 2014 Tanca et al.; licensee BioMed central Ltd.|
|Deposited On:||19 Dec 2014 13:54|
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