Ultra-sensitivity metaproteomics redefines the gut dark metaproteome, uncovering host-microbiome interactions and drug targets in intestinal inflammatory diseases.

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 Crohns patients and presented the concept of a "druggable metaproteome". Among the drug-protein interactions discovered are treatments for intestinal inflammatory diseases, showcasing uMetaPs potential for disease diagnostics and data-driven drug repurposing strategies.