pXg: Comprehensive and Reliable Identification of MHC-I Antigens Using De Novo Peptide Sequencing and...

De novo peptide sequencing enables the identification of a broad repertoire of major histocompatibility complex class I-associated peptides, providing a valuable source of potential immunotherapeutic targets in cancer. Nevertheless, its practical utility remains limited by the absence of robust methods for confidently determining peptide-spectrum matches and interpreting their proteogenomic information. This chapter presents the pXg workflow, designed to comprehensively and reliably identify peptides derived from de novo sequencing in mass spectrometry-based immunopeptidomics, integrated with matched RNA-seq data. We guide the reader through the use of pXg, covering the rescoring of peptide-spectrum matches and the estimation of a false discovery rate from the popular de novo sequencing tool Casanovo. This ultimately yields rich proteogenomic insights into the identified peptides.