Thunder-DDA-PASEF enables high-coverage immunopeptidomics and identifies HLA class-I presented SarsCov-2 spike protein epitopes

Abstract Human leukocyte antigen (HLA) class I peptide ligands (HLAIps) are key targets for developing vaccines and immunotherapies against infectious pathogens or cancer cells. Identifying HLAIps is challenging due to their high diversity, low abundance, and patient-specific profiles. Here, we developed a highly sensitive method for identifying HLAIps using liquid chromatography-ion mobility-tandem mass spectrometry (LC-IMS-MS/MS). The optimized method, Thunder-DDA-PASEF, semi-selectively fragments HLAIps based on their IMS and m/z, thus increasing the coverage of immunopeptidomics analyses. Thunder-DDA-PASEF includes singly-charged peptides, which contributes to more than 35% of the HLAIp identifications. Combined with MS2Rescore, Thunder-DDA-PASEF improved ligandome coverage by 150% compared to the original-DDA-PASEF method, and enabled in-depth profiling of HLAIps from two human cell lines, JY and Raji, transfected to express the SARS-CoV-2 spike protein. We identified seventeen spike protein HLAIps, thirteen of which had been reported to elicit immune responses in human patients.