A facile immunopeptidomics workflow for capturing the HLA-I ligandome with PEAKS XPro

Identifying antigens displayed specifically on tumour cell surfaces by human leukocyte antigen (HLA) proteins is important for the development of immunotherapies and cancer vaccines. The difficulty in capturing an HLA ligandome stems from the fact that many HLA ligands are derived from splicing events or contain mutations, hindering their identification in a standard database search. To address this challenge, we developed an immunopeptidomics workflow with PEAKS XPro that uses de novo sequencing to uncover such peptides and identifies mutations for neoantigen discovery. We demonstrate the utility of this workflow by re-analyzing HLA-I ligandome datasets and reveal a vast diversity in peptide sequences among clones derived from a colorectal cancer tumour. Over 8000 peptides predicted to bind HLA-I molecules were identified by de novo sequencing only (not found in the UniProt database) and make up over 50% of identified peptides from each sample. Lastly, tumour-specific mutations and consensus sequence motif characteristics are defined. This workflow is widely applicable to any immunopeptidomic mass spectrometry dataset and does not require custom database generation for neoantigen discovery.