Expanding the human metaproteome: enhancing small open reading frame (smORF) prediction and ultra-deep detection of smORF-encoded proteins in the gut microbiome

Small open reading frames (smORFs), which encode proteins under 100 amino acids, represent an underexplored dimension of the human gut microbiome, despite growing evidence of their essential biological roles. Due to small size and poor annotation, smORFs are typically excluded from metagenomic/metaproteomic analyses. Here, we present a high-resolution multi-omic workflow that integrates smORF prediction into metaproteome searches and enables ultra-deep detection of smORF-encoded proteins (SEPs), without experimental size-based enrichment, utilizing state-of-the-art mass spectrometry instrumentation. Applied to human gut microbiomes, this approach provides the most comprehensive SEP detection to date, allowing identification of more than 30,000 SEPs in the metaproteome, alongside the measurements of the larger proteins. Our multi-omics integrated strategy is not only critical for advancing human metaproteome research but also provides a generalizable strategy for comprehensive SEP discovery across diverse microbial ecosystems, thereby greatly expanding the accessible proteomic landscape that has been previously hidden.