Abstract
Small proteins (SPs, ≤50 aa) are often overlooked in genomics. We conducted the first systematic analysis of prokaryotic SPs across the full ocean-depth gradient. From 433,311 short open reading frames (sORFs) predicted from 71 western Pacific metagenomes, we identified 193,281 SP clusters. Filtration yielded 75,581 prevalent SPs, including 4,307 high-confidence clusters (RfSPs). Notably, 87.09% of RfSPs lacked non-marine homologs, and ∼70% contained unknown domains. While most (65.57%) were phylum-specific, twelve were distributed across ≥5 phyla, and some were prophage-associated. Geographically, twenty-three core RfSPs were universally present. Co-occurrence analysis revealed that interacting RfSPs typically originated from the same or adjacent zones. Finally, we confirmed the transcription of 8.20% RfSP clusters in deep-sea metatranscriptomes. The zone-specific transcription of certain RfSPs suggests adaptive functions, such as stress response and molecular chaperoning, in distinct marine environments. Our study reveals SPs as a critical strategy for prokaryotic adaptation to deep-sea stressors.