PEXMap: A proteogenomic method for exon and isoform level mapping of mass spectrometry derived peptides

Alternative splicing (AS) expands transcriptome and proteome diversity by differentially combining exons or their splice variants. Although RNA-seq studies have uncovered transcriptomic variability, understanding the corresponding protein-level diversity remains limited. Mass spectrometry-based proteomics provides protein-level insights through MS/MS peptide annotations, which are mostly linked to gene/transcript or UniProt identifiers. However, tracing them to specific isoforms remains challenging due to the lack of exon mapping or inconsistent annotations. We developed PEXMap (PeptideEXonMapper), a k-mer-based proteogenomic framework that systematically maps MS/MS peptides to genes, transcripts, exons, or exon-exon junctions by exact matching of unique 8-mers derived from MS/MS peptides to those in reference databases from exon-resolved isoforms. Comparing PEXMap mappings of human proteome from PeptideAtlas showed annotation concordance with it. Applying PEXMap to liver and pancreas proteomes, we identified tissue-specific isoform expression and, similarly, annotated the cancer proteome. PEXMap reliable mappings could provide insights into role of AS in shaping proteomes across tissues and disease states. Source code is publicly available for download at GitHub: https://github.com/deepanshicbg/PEXMap and supported on Linux.