Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines.
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.
Antwi
Kwasi
K
School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States.
Hanavan
Paul D
PD
Myers
Cheryl E
CE
Ruiz
Yvette W
YW
Thompson
Eric J
EJ
Lake
Douglas F
DF
D000925:Anticoagulants / Q000494:pharmacology; D001943:Breast Neoplasms / Q000276:immunology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D019343:Citric Acid / Q000494:pharmacology; D005260:Female; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010190:Pancreatic Neoplasms / Q000276:immunology*; D010206:Papain / Q000494:pharmacology; D010446:Peptide Fragments / Q000276:immunology* / Q000302:isolation & purification; D020543:Proteome / Q000276:immunology*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D000925:Anticoagulants; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D020543:Proteome; D019343:Citric Acid; D010206:Papain