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 journalArticle

18 Citations (Scopus)

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

Fingerprint

Pancreatic Neoplasms
Proteomics
Breast Neoplasms
Cell Line
Peptides
Amino Acids
Immune System
Neoplasms
Acids
Antibody Affinity
Papain
Tumor Cell Line
Citric Acid
Names
Buffers

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines. / Antwi, Kwasi; Hanavan, Paul D.; Myers, Cheryl E.; Ruiz, Yvette W.; Thompson, Eric J.; Lake, Douglas.

In: Molecular Immunology, Vol. 46, No. 15, 09.2009, p. 2931-2937.

Research output: Contribution to journalArticle

Antwi, Kwasi ; Hanavan, Paul D. ; Myers, Cheryl E. ; Ruiz, Yvette W. ; Thompson, Eric J. ; Lake, Douglas. / Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines. In: Molecular Immunology. 2009 ; Vol. 46, No. 15. pp. 2931-2937.
@article{e1ba62c7974147b895b39d5893d2a692,
title = "Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines",
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.",
keywords = "Cell line, Immune system, Liquid chromatography, Major histocompatibility complex, Mass spectrometry, MHC-refolding assay, Peptides",
author = "Kwasi Antwi and Hanavan, {Paul D.} and Myers, {Cheryl E.} and Ruiz, {Yvette W.} and Thompson, {Eric J.} and Douglas Lake",
year = "2009",
month = "9",
doi = "10.1016/j.molimm.2009.06.021",
language = "English (US)",
volume = "46",
pages = "2931--2937",
journal = "Molecular Immunology",
issn = "0161-5890",
publisher = "Elsevier Limited",
number = "15",

}

TY - JOUR

T1 - Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines

AU - Antwi, Kwasi

AU - Hanavan, Paul D.

AU - Myers, Cheryl E.

AU - Ruiz, Yvette W.

AU - Thompson, Eric J.

AU - Lake, Douglas

PY - 2009/9

Y1 - 2009/9

N2 - 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.

AB - 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.

KW - Cell line

KW - Immune system

KW - Liquid chromatography

KW - Major histocompatibility complex

KW - Mass spectrometry

KW - MHC-refolding assay

KW - Peptides

UR - http://www.scopus.com/inward/record.url?scp=68749110478&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=68749110478&partnerID=8YFLogxK

U2 - 10.1016/j.molimm.2009.06.021

DO - 10.1016/j.molimm.2009.06.021

M3 - Article

C2 - 19615748

AN - SCOPUS:68749110478

VL - 46

SP - 2931

EP - 2937

JO - Molecular Immunology

JF - Molecular Immunology

SN - 0161-5890

IS - 15

ER -