Wu, Q.; Ning, Z.; Zhang, A.; Cheng, K.; Figeys, D.

Taxonomic interpretation of metaproteomic peptides remains difficult because many peptide sequences are present in proteins from different organisms, reducing taxonomic specificity. Current peptide-centric workflows can report taxonomic summaries or taxon level confidence scores, but they do not provide formal statistical evidence that a taxon is present in the microbiome. Here we present MetaUmbra, a tool that derives genome-level statistical significance values from identified peptides. MetaUmbra builds theoretical peptide lists by in silico digestion of the taxon specific proteins and matches observed peptides against these references. It then combines a conservative significance estimate from unique peptides with a Monte Carlo based p-value for shared peptide evidence estimated under an empirical null model. In the defined community benchmark SIHUMIx, MetaUmbra identified the expected genomes without introducing false-positive genomes after embedding the SIHUMIx genomes in a large gut reference background. In the single strain benchmark Mix24X, all expected genomes were identified with the best statistical significances even after near neighbor and full background expansion. In a hamster gut genome panel, MetaUmbra further preserved an interpretable ranking of candidate genomes in a dense real-data setting. Together, these results show that MetaUmbra can statistically identify the presence of specific microbes in a complex microbiome while maintaining low false-positive calls. MetaUmbra therefore provides a practical framework for converting peptide evidence into genome-level statistical inference in metaproteomics.