pXg: Comprehensive Identification of Noncanonical MHC-I–Associated Peptides From De Novo Peptide Sequencing Using RNA-Seq Reads

Discovering noncanonical peptides has been a common application of proteogenomics. Recent studies suggest that certain noncanonical peptides, known as noncanonical major histocompatibility complex-I (MHC-I)-associated peptides (ncMAPs), that bind to MHC-I may make good immunotherapeutic targets. De novo peptide sequencing is a great way to find ncMAPs since it can detect peptide sequences from their tandem mass spectra without using any sequence databases. However, this strategy has not been widely applied for ncMAP identification because there is not a good way to estimate its false-positive rates. In order to completely and accurately identify immunopeptides using de novo peptide sequencing, we describe a unique pipeline called proteomics X genomics. In contrast to current pipelines, it makes use of genomic data, RNA-Seq abundance and sequencing quality, in addition to proteomic features to increase the sensitivity and specificity of peptide identification. We show that the peptide-spectrum match quality and genetic traits have a clear relationship, showing that they can be utilized to evaluate peptide-spectrum matches. From 10 samples, we found 24,449 canonical MHC-I–associated peptides and 956 ncMAPs by using a target-decoy competition. Three hundred eighty-seven ncMAPs and 1611 canonical MHC-I–associated peptides were new identifications that had not yet been published. We discovered 11 ncMAPs produced from a squirrel monkey retrovirus in human cell lines in addition to the two ncMAPs originating from a complementarity determining region 3 in an antibody thanks to the unrestricted search space assumed by de novo sequencing. These entirely new identifications show that proteomics X genomics can make the most of de novo peptide sequencing’s advantages and its potential use in the search for new immunotherapeutic targets. [Display omitted] •We implemented pXg to confidently identify de novo peptides using RNA-Seq reads.•pXg incorporates proteogenomic features into Percolator in immunopeptidomics.•RNA-Seq quantity/quality has a clear relationship with PSM quality.•pXg enables identifying every potential immunopeptidome including viral proteins. Identification of noncanonical peptides is an essential task for discovery novel immunotherapeutic targets. Due to the limited search space by prior knowledge, database searches struggle to identify comprehensive noncanonical peptides. In order to completely and accurately identify immunopeptides using both de novo