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

Kwasi Antwi, Paul D. Hanavan, Cheryl E. Myers, Yvette W. Ruiz, Eric J. Thompson, Douglas Lake

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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 25 min at pH 3.0. Panc-1 remained >90% intact after 45 min 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 45 min 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.

Original languageEnglish (US)
Pages (from-to)2931-2937
Number of pages7
JournalMolecular Immunology
Volume46
Issue number15
DOIs
StatePublished - Sep 2009

Keywords

  • Cell line
  • Immune system
  • Liquid chromatography
  • MHC-refolding assay
  • Major histocompatibility complex
  • Mass spectrometry
  • Peptides

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

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