The amount of public proteomics data is rapidly increasing but there is no standardized format to describe the sample metadata and their relationship with the dataset files in a way that fully supports their understanding or reanalysis. Here we propose to develop the transcriptomics data format MAGE-TAB into a standard representation for proteomics sample metadata. We implement MAGE-TAB-Proteomics in a crowdsourcing project to manually curate over 200 public datasets. We also describe tools and libraries to validate and submit sample metadata-related information to the PRIDE repository. We expect that these developments will improve the reproducibility and facilitate the reanalysis and integration of public proteomics datasets.

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.

Introduction: The study of microbial communities based on the combined analysis of genomic and proteomic data - called metaproteogenomics - has gained increased research attention in recent years. This relatively young field aims to elucidate the functional and taxonomic interplay of proteins in microbiomes and its implications on human health and the environment. Areas covered: This article reviews bioinformatics methods and software tools dedicated to the analysis of data from metaproteomics and metaproteogenomics experiments. In particular, it focuses on the creation of tailored protein sequence databases, on the optimal use of database search algorithms including methods of error rate estimation, and finally on taxonomic and functional annotation of peptide and protein identifications. Expert opinion: Recently, various promising strategies and software tools have been proposed for handling typical data analysis issues in metaproteomics. However, severe challenges remain that are highlighted and discussed in this article; these include: (i) robust false positive assessment of peptide and protein identifications, (ii) complex protein inference against a background of highly redundant data, (iii) taxonomic and functional post-processing of identification data, and finally, (iv) the assessment and provision of metrics and tools for quantitative analysis.

Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. The international metaproteomics community is delighted to announce the launch of the Metaproteomics Initiative (www.metaproteomics.org), the goal of which is to promote dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research. The Initiative aims to be the central information hub and open meeting place where newcomers and experts interact to communicate, standardize, and accelerate experimental and bioinformatic methodologies in this field. We invite the entire microbiome community to join and discuss potential synergies at the interfaces with other disciplines, and to collectively promote innovative approaches to gain deeper insights into microbiome functions and dynamics.

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. By integrating metaproteomics with other omics disciplines, researchers can gain a comprehensive understanding of microbial ecology, interactions, and functional dynamics. This review, developed by the Metaproteomics Initiative (www.metaproteomics.org), serves as a practical guide for both microbiome and proteomics researchers, presenting key principles, state‐of‐the‐art methodologies, and analytical workflows essential to metaproteomics. Topics covered include experimental design, sample preparation, mass spectrometry techniques, data analysis strategies, and statistical approaches.