Highlights d In vivo screen for fibers targeting specific human gut taxa in a defined community d Proteomics and forward genetics identify bioactive nutrients and their utilization d Interspecies competition controls the outcome of fiberbased microbiota manipulation d Artificial food particles as biosensors of community-wide glycan degradation

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Highlights d Fructoselysine (FL) is a common Maillard reaction product (MRP) in whey protein d FL selectively increases fitness of Collinsella intestinalis in gnotobiotic mice d C. intestinalis metabolizes FL via regulated transcription of a FL utilization locus d Gut bacteria may affect food safety through MRP degradation to harmless products

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-lab comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, lab-assembled human intestinal model and a human fecal sample. We observed that variability at the peptide level was predominantly due to wet-lab workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-lab studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Previous bioinformatic analyses of metagenomic data have indicated that bacteriophages can use genetic codes different from those of their host bacteria. In particular, reassignment of stop codon TAG to glutamine (a variation known as ‘genetic code 15’) has been predicted. Here, we use LC-MS/MS-based metaproteomics of human fecal samples to provide experimental evidence of the use of genetic code 15 in two crAss-like phages. Furthermore, the proteomic data from several phage structural proteins supports the reassignment of the TAG stop codon to glutamine late in the phage infection cycle. Thus, our work experimentally validates the expression of genetic code 15 in human microbiome phages.