Abstract
Genome sequences encode the full blueprint of life. However, functional annotation, especially of short open reading frames (sORFs), remains uncertain. Many small proteins are likely overlooked by standard algorithms. Proteomics using LC-MS/MS enables protein detection but depends on existing databases, which underrepresent sORFs. We developed a proteogenomic workflow integrating MS/MS data with genome-wide ORF predictions to detect novel small proteins. Applied to Staphylococcus aureus and other bacteria, it revealed numerous previously unannotated sORFs, expanding our understanding of microbial proteomes.