SAPrIm, A Semi-Automated Protocol for mid-throughput Immunopeptidomics

Human leukocyte antigen (HLA) molecules play a crucial role in the development of adaptive immune responses and therefore have been of major interest in the development of immunotherapies such as cancer vaccines and chimeric antigen receptor (CAR) T cells. Hence, a comprehensive understanding and profiling of the immunopeptidome is required to foster growth for these personalised solutions. We herein describe a novel immunopeptidomics workflow involving the Kingfisher platform (Thermo Scientific) to isolate immunopeptidomes with anti-HLA antibodies coupled to a proprietary hyper-porous protein A microparticle (MagReSyn®), and a modified variable window data independent acquisition (DIA) method. Using the workflow, we were able to identify ~1500 peptides in as low as 5e6 cells. Overall, the robustness of this workflow will be very important for the future of the immunopeptidome profiling, especially for smaller sample types, and will be a useful tool for the development of immunotherapies and other precision medicine approaches.