timsTOF mass spectrometry-based immunopeptidomics refines tumor antigen identification

Abstract T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the de novo implementation of ion mobility separation-based timsTOF MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. A direct comparison of timsTOF-based with state-of-the-art immunopeptidomics using orbitrap technology showed significantly increased HLA peptide identifications from benign and malignant primary samples of solid tissue and hematological origin. First application of timsTOF-based immunopeptidomics for tumor antigen discovery enabled (i) the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides from 94 primary benign tissue samples, (ii) the refinement of previously described tumor antigens, and (iii) the identification of a vast array of novel tumor antigens, comprising low abundant neoepitopes, that might serve as targets for future cancer immunotherapy development.