Ultra-sensitive metaproteomics (uMetaP) redefines the dark field of metaproteome, enables single-bacterium resolution, and discovers hidden functions in the gut microbiome

Metaproteomics uniquely characterizes host-microbiome interactions. However, most species detected by metagenomics remain hidden to metaproteomics due to sensitivity limits. We present a novel ultra-sensitive metaproteomic solution (uMetaP) that, for the first time, reaches full-length 16S rRNA taxonomic depth and can simultaneously decipher functional features. Querying the mouse gut microbiome, uMetaP achieved unprecedented performance in key metrics like protein groups (47925) alongside taxonomic (220 species) and functional annotations (223 KEGG pathways) - all within 30-min analysis time and with high reproducibility, sensitivity, and quantitative precision. uMetaP revealed previously unidentified proteins of unknown functions, small proteins, and potentially new natural antibiotics. Leveraging the extreme sensitivity of uMetaP and SILAC-labelled bacteria, we revealed the true limit of detection and quantification for the "dark" metaproteome of the mouse gut. Moreover, using a two-bacteria proteome mix, we demonstrated single-bacterium resolution (500 fg) with exceptional quantification precision and accuracy. From deciphering the interplay of billions of microorganisms with the host to exploring microbial heterogeneity, uMetaP represents a quantum leap in metaproteomics. Taken together, uMetaP will open new avenues for our understanding of the microbial world and its connection to health and disease.