High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera

Proteomics. 2016 May;16(10):1570-80. doi: 10.1002/pmic.201500445.


The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell-driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA-bound peptides has typically required billions of cells, still resulting in relatively few high-confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid-eluted HLA peptides after purification with the pan-reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high-confidence peptides could be identified, including melanoma-associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology-based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.

Keywords: Biomedicine; HLA class I; Immunopeptidomics; Melanoma; sHLA tumor-associated epitopes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Antigens, Neoplasm / blood
  • Biomarkers, Tumor / blood*
  • Biomarkers, Tumor / isolation & purification
  • Case-Control Studies
  • Consensus Sequence
  • HEK293 Cells
  • HL-60 Cells
  • Histocompatibility Antigens Class I / physiology*
  • Humans
  • Melanoma / blood*
  • Peptides / blood*
  • Peptides / isolation & purification
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Spondylitis, Ankylosing / blood


  • Antigens, Neoplasm
  • Biomarkers, Tumor
  • Histocompatibility Antigens Class I
  • Peptides