MetaPilot: genome-aware adaptive search-space refinement for unified DDA and DIA metaproteomics

Metaproteomic peptide identification is constrained by the structure and size of the protein search space. Pooled gene catalogues provide coverage but obscure genome-level evidence, and current workflows for data-dependent (DDA) and data-independent (DIA) acquisition diverge in their database strategies. We present MetaPilot, a genome-aware workflow that uses conserved marker-protein evidence to rank candidate genomes from MGnify catalogues and construct adaptive, sample-specific search spaces. Applied to paired DDA/DIA datasets of defined mixtures and fecal samples, MetaPilot adapted genome selection to community complexity and reproduced published peptide evidence while expanding the detectable peptide space. In DDA-independent reanalysis of Orbitrap human gut DIA data, MetaPilot identified 24.4% more peptides than the published DDA-derived library and 2.06-fold more than the matched DDA-assisted DIA search. On timsTOF DIA-PASEF mouse intestinal data, it outperformed uMetaP by 41.8~119.7%, enabling genome-resolved functional interpretation without DDA-PASEF input.