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. 2013 Nov;12(11):3310-8.
doi: 10.1074/mcp.M113.029967. Epub 2013 Aug 27.

Host-centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota

Joshua S Lichtman  1 , Angela Marcobal, Justin L Sonnenburg, Joshua E Elias
Affiliations

Host-centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota

Joshua S Lichtman et al. Mol Cell Proteomics. 2013 Nov.

Abstract

The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota ('humanized'), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a "core" stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool.

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Figures

Fig. 1.
Fig. 1.
A novel method for the analysis of the mammalian gut proteome. A, In this workflow, the resulting supernatant from differential centrifugation is fractionated with C-4 reverse phase solid-phase extraction to enrich for proteins and peptides. The resulting fractions are digested with trypsin, de-salted and applied to reverse-phase LC-MS/MS to study the host contribution to the gut proteome. The resulting data can then be applied to univariate and multivariate analyses. B, The distribution of peptide-spectral matches between fully-tryptic and semitryptic peptides in germ-free and conventional mice indicating the importance for utilizing semitryptic parameters when searching this data. C, The frequency of identification across biological replicates in germ-free and conventional mice. Comparing parts b and c, there are more spectra per unique peptide in the fully-tryptic category but fewer total unique peptides, indicating that the fully-tryptic ones are more abundant and less diverse.
Fig. 2.
Fig. 2.
The effect of microbial complexity on host gut protein expression in mice. Mice were colonized with the microbial communities detailed in Table I and single fecal samples were subjected to the described workflow. A, Principal component analysis of spectral counts for the 2529 proteins identified across the 22 mice. B, The frequency of protein identification between biological replicates within a colonization state shows increased protein diversity in mice with a complex microbiota. C, Expression of functionally related proteins across colonization states. y-axis: % total expression (normalized spectral counts) in indicated colonization state compared with the sum of all states, error bars: Mean + S.E.
Fig. 3.
Fig. 3.
Interindividual variation exceeds variation within an individual over time. Principal component analysis of the fecal proteomes of two germ free and two conventional mice sampled for 3 days.
Fig. 4.
Fig. 4.
Application of secreted proteome enrichment method to human fecal samples. A, Functional annotation enrichment of the 244 human proteins over the human proteome compared with the enrichment found in a recent gut proteome analysis (10). B, GO term enrichment calculated using DAVID for the proteins found in the core versus all detected proteins. C, Hierarchical clustering of the normalized fold-change protein expression compared with the average of the three individuals.
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