Abstract
BackgroundThe 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.ResultsThis 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-014-0049-2) contains supplementary material, which is available to authorized users.
Highlights
The massive characterization of host-associated and environmental microbial communities has represented a real breakthrough in the life sciences in the last years
We present an optimized analytical pipeline for analysis and characterization of metaproteomes, which comprises bead-beating/freeze-thawing for protein extraction, filter-aided sample preparation (FASP) for cleanup and digestion, and single-run nanoLC-tandem mass spectrometry (MS/mass spectrometry (MS)) for peptide separation and identification
Overview of pipeline steps and study design The pipeline for metaproteome analysis presented in this work, as detailed below in the “Methods” section and illustrated in Figure 2A, consists of three main steps: i) proteins are extracted from microbial community samples by heating in SDS-based buffer followed by bead-beating/ freeze-thawing steps; ii) protein extracts are cleaned up and digested on-filter according to the FASP procedure (8 h to overnight); and iii) peptide mixtures are subjected to single-run liquid chromatography (LC)-MS/MS analysis using an 8-h gradient
Summary
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 enables the transition from assessing the genomic potential to measuring the functional expression of a microbiome. Given the relative youth of metaproteomics, as well as the huge variability among microbial community samples and within community members in terms of biochemical features and structural complexity, significant research efforts are still required to improve, optimize, and standardize sample preparation workflows for metaproteome analysis. Since Gram-positive bacteria, Gram-negative bacteria, and fungi show tremendous structural differences and diverse extents of susceptibility to each protein extraction method, an optimized protocol should maximize extraction yield, avoiding a selective depletion of species with higher resistance to lysis
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