Mass spectrometry-based immunopeptidomics is the gold standard technique for identifying peptides in complex with human leukocyte antigens (HLA). These HLA bound peptides are scrutinised by the adaptive immune system where they can illicit cytotoxic T cell responses. These peptides provide insights into the dynamic intracellular environment so that each pathophysiological condition contains a unique peptide repertoire. This enables the discovery of disease-specific antigens that can inform vaccine and immunotherapy design and development. However, current immunopeptidomic workflows require large input materials and labour-intensive, multi-day protocols, limiting their feasibility for clinical and preclinical applications at a large scale.
To address this challenge, we present our semi-automated, high-throughput immunopeptidomic workflow with the ability to process up to 96 samples in a single day. Utilising the KingFisher Apex and leveraging the orbitrap Astral mass spectrometer immunopeptide enrichment and identification are performed at unprecedented depth. Using clinical biopsy material from melanoma, sarcoma, brain and colorectal cancer >95% of peptides identified fit canonical length binding distribution of HLA alleles (8-12mer peptides) with peptide predicted binding ranging from 80-90% of peptides. We identify a vast repertoire of actionable antigens from as little as 3mgs of tissue in osteosarcoma with ~29,000 peptides, whilst in melanoma we observe from 100ul of plasma (soluble HLA) ~7,000 peptides. Importantly, we encounter tumour associated antigens across multiple cancer types such as MAGE, P53 and other cancer testis antigens providing an avenue for off the shelf cancer vaccines with common HLA allotypes. Taken together high resolution immunopeptidomics offers a unique avenue into exploring actionable cell surface targets presented on the surface of tumours. Using state of the art instrumentation and techniques we can identify cancer restricted peptides across low input tumour types for T cell mediated immunotherapy techniques as well as off the shelf cancer vaccine approaches.