pAnno: a comprehensive, precise, and fast proteogenomic workflow for the discovery of novel coding regions

Proteogenomics is a transformative approach for deciphering novel coding regions through integration of genomic, transcriptomic, and proteomic data. Here, we present pAnno, an end-to-end workflow designed to uncover hidden protein-coding elements with high precision and efficiency. pAnno generates customized protein databases by integrating multi-omic data, employs a multi-stage iterative open search strategy, and incorporates an efficient peptide-to-coding sequence mapping algorithm. Despite a 50-fold increase in database size, pAnno maintains high sensitivity and accuracy in peptide identification and achieves genomic localization of novel events with only $\sim$ 3% additional processing time, delivering unprecedented resolution and speed in proteogenomic analysis. By detecting splicing, mutations, and novel protein isoforms, pAnno supports various downstream applications and reveals overlooked events, identifying 1.73 $\times$ more novel proteins in Pyrus and 34 $\times$ more non-canonical HLA peptides in lung cancer. These capabilities position pAnno as a gold-standard proteogenomic workflow, excelling in non-canonical coding discovery and large-scale database processing.