Mass Spectrometry Based Immunopeptidomics for the Discovery of Cancer Neoantigens

Recent data indicate that endogenous mutated cancer proteins can be processed and presented as HLA binding peptides, leading to their recognition in vivo as "non-self." Targeting such neoantigens would enable immune cells to distinguish between normal and cancerous cells, avoiding the risk of autoimmunity. So far, discovery of such neoantigens relies mainly on prediction-based interrogation of the "mutanome" using genomic information as input, followed by highly laborious and time-consuming T cell screening assays. Currently, mass spectrometry is the only unbiased methodology to comprehensively interrogate the naturally presented repertoire of HLA binding peptides, including peptides derived from tumor-associated antigens and post-translational modified peptides. This chapter describes a detailed protocol for in-depth and accurate mass spectrometry based immunopeptidomics, enabling the direct identification of tissue-derived neoantigens extracted from human tumors.