Comparative metaproteomics and diversity analysis of human intestinal microbiota testifies for its temporal stability and expression of core functions

The human intestinal tract is colonized by microbial communities that show a subject-specific composition and a high-level temporal stability in healthy adults. To determine whether this is reflected at the functional level, we compared the faecal metaproteomes of healthy subjects over time using a novel high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry. The developed robust metaproteomics workflow and identification pipeline was used to study the composition and temporal stability of the intestinal metaproteome using faecal samples collected from 3 healthy subjects over a period of six to twelve months. The same samples were also subjected to DNA extraction and analysed for their microbial composition and diversity using the Human Intestinal Tract Chip, a validated phylogenetic microarray. Using metagenome and single genome sequence data out of the thousands of mass spectra generated per sample, approximately 1,000 peptides per sample were identified. Our results indicate that the faecal metaproteome is subject-specific and stable during a one-year period. A stable common core of approximately 1,000 proteins could be recognized in each of the subjects, indicating a common functional core that is mainly involved in carbohydrate transport and degradation. Additionally, a variety of surface proteins could be identified, including potential microbes-host interacting components such as flagellins and pili. Altogether, we observed a highly comparable subject-specific clustering of the metaproteomic and phylogenetic profiles, indicating that the distinct microbial activity is reflected by the individual composition