Mass Spectrometry Based Immunopeptidomics for the Discovery of Cancer Neoantigens

Methods Mol Biol. 2018;1719:209-221. doi: 10.1007/978-1-4939-7537-2_14.


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.

Keywords: Cancer immunotherapy; HLA binding peptides; Immunoaffinity purification; Immunopeptidomics; Mass spectrometry; Neoantigens.

MeSH terms

  • Antigens, Neoplasm / analysis
  • Antigens, Neoplasm / immunology
  • Antigens, Neoplasm / metabolism*
  • Chromatography, Affinity / methods*
  • Histocompatibility Antigens Class I / analysis
  • Histocompatibility Antigens Class I / immunology
  • Histocompatibility Antigens Class I / metabolism*
  • Histocompatibility Antigens Class II / analysis
  • Histocompatibility Antigens Class II / immunology
  • Histocompatibility Antigens Class II / metabolism*
  • Humans
  • Mass Spectrometry / methods*
  • Neoplasms / immunology
  • Neoplasms / metabolism*
  • Peptide Fragments / analysis
  • Peptide Fragments / immunology
  • Peptide Fragments / metabolism*


  • Antigens, Neoplasm
  • Histocompatibility Antigens Class I
  • Histocompatibility Antigens Class II
  • Peptide Fragments