Advancements in high-resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13,829 peptides were identified; 83-87% of these were 8-11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA-type binding prediction for 10,078 9/10 mer peptides assigned 88-95% to a patient-specific HLA subtype, revealing a disparity in strength of predicted binding. HLA-B*27-specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.
Keywords: MHC class I; antigen; epitope; mass spectrometry; melanoma; peptide; proteome.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.