Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines

Mol Immunol. 2009 Sep;46(15):2931-7. doi: 10.1016/j.molimm.2009.06.021. Epub 2009 Jul 16.

Abstract

Peptides bound to cell surface MHC class I molecules allow the immune system to recognize intracellular pathogens and tumor-derived peptides. Our goal was to learn what the immune system "sees" on the surfaces of tumor cells by acid-eluting peptides from HLA molecules for extended time periods. We determined how long peptides would continue to elute over time from a pancreatic tumor cell line, Panc-1, and a breast cancer cell line, MCF-7, at pH 3.0 in citrate buffer while monitoring viability. Both cell lines demonstrated greater than 90% viability after 25min at pH 3.0. Panc-1 remained >90% intact after 45min at pH 3.0. Acid eluted peptide sequences were identified using LC-MS/MS and searching the NCBI refseq database. The total number of peptides eluted peaked between 40 and 45min for Panc-1, but continued to increase over time from MCF-7. A total of 131 peptides were identified from Panc-1 while 101 peptides were identified from MCF-7 elutions. Two classes of peptides were eluted: (1) 8-10 amino acid peptides fitting the HLA-binding motifs of each cell line, and (2) peptides longer than 10 amino acids containing HLA-binding motifs of each cell line. W6/32 antibody affinity purification of intact MHC molecules after papain cleavage of MHC class I from tumor cell surfaces also indicated that peptides longer than 10 amino acids bind to class I proteins. A peptide-MHC-refolding assay further substantiated the binding of longer peptides to HLA-A*0201. Our findings provide sequences and gene names of peptides presented by MHC class I molecules from common pancreas and breast cancer cell lines. We utilized a novel refolding assay to demonstrate that peptides longer than the canonical 8-10 amino acids commonly bind in MHC class I cell surface molecules.

MeSH terms

  • Anticoagulants / pharmacology
  • Breast Neoplasms / immunology*
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Chromatography, High Pressure Liquid
  • Citric Acid / pharmacology
  • Female
  • Histocompatibility Antigens Class I / immunology*
  • Histocompatibility Antigens Class I / isolation & purification
  • Humans
  • Hydrogen-Ion Concentration
  • Pancreatic Neoplasms / immunology*
  • Papain / pharmacology
  • Peptide Fragments / immunology*
  • Peptide Fragments / isolation & purification
  • Proteome / immunology*
  • Proteomics
  • Tandem Mass Spectrometry

Substances

  • Anticoagulants
  • Histocompatibility Antigens Class I
  • Peptide Fragments
  • Proteome
  • Citric Acid
  • Papain