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 an automated, high-throughput immunopeptidomic workflow with the ability to process up to 96 samples in a single day. Utilising the KingFisher Apex liquid handler robot this protocol streamlines immunopeptide antibody-based enrichment, significantly reducing manual intervention while enhancing reproducibility. Building upon our previously published SAPrIm method where we showcased that our variable DIA window approach can reproducibly identify and quantify over 13,000 HLA peptides using 5e7 cells. This method is scalable down to 500,000 cells, allowing for low input immunopeptidomics for clinical or rare sample types. By harnessing high-resolution, state-of-the-art mass spectrometers and leveraging expertise and method development at the Monash Proteomics and Metabolomics Platform, this platform promises to deliver unprecedented depth and accuracy in immunopeptidomic analysis. This open-access resource will be available to both academic and industry researchers facilitating a range of large-scale clinical and translational research applications.