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The gut microbiome is a complex ecosystem with significant inter-individual variability determined by hundreds of low-abundant species as revealed by genomic methods. Functional redundancy demands direct quantification of microbial biological functions to understand their influence on host physiology. This functional landscape remains unexplored due to limited sensitivity in metaproteomics methods. We present uMetaP, an ultra-sensitive metaproteomic solution combining advanced LC-MS technologies with a novel FDR-controlled de novo strategy. uMetaP improves the taxonomic detection limit of the gut "dark metaproteome" by 5,000-fold with exceptional quantification precision and accuracy. In a mouse model of colonic injury, uMetaP extended metagenomics findings and identified host functions and microbial metabolic networks linked to disease. We obtained orthogonal validation using transcriptomic data from biopsies of 204 Crohn's patients and presented the concept of a "druggable metaproteome". Among the drug-protein interactions discovered are treatments for intestinal inflammatory diseases, showcasing uMetaP's potential for disease diagnostics and data-driven drug repurposing strategies.
Competing Interest Statement
M.S. received research awards and travel support from the German Pain Society (DGSS) both of which were sponsored by Astellas Pharma GmbH (Germany). MS received research awards from the Austrian Pain Society. MS received a one-time consulting honorarium from Grunenthal GmbH (Germany). None of these sources influenced the content of this study, and MS declares no conflict of interest. D.G.V. and M.S. have an ongoing scientific collaboration with Bruker (Center of Excellence for Metaproteomics University of Vienna - Bruker), however, this collaboration did not influence the content of the manuscript. D.G.V, F.X., M.B, R.K., A.B., D.A., D.H., and M.S. declare that they have no conflicts of interest. C.K., J.K., and T.S. are employees of Bruker Daltonics GmbH & Co. Q.L. and B.M. are employees of Rapid Novor.
Footnotes
* This version of the manuscript has been revised to include new experiments.