High-throughput MS-based immunopeptidomics

Comprehensive knowledge of the HLA class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their extraction for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential extraction of HLA-I and -II peptides from up to 96 samples in a plate format from cell lines or tissues. Our methodology drastically reduces sample-handling and can be completed within six hours. We report identification and quantification of thousands of peptides with excellent reproducibility (Pearson correlations reaching 0.98 and 0.97, class I and II, respectively). Due to improved sensitivity, as many as 1,846 HLA-I and 2,633 HLA-II peptides were accurately identified from only 107 B-cells. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma. This straightforward method is applicable for basic and clinical applications.