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 language | English (US) |
|---|---|
| Pages (from-to) | 2931-2937 |
| Number of pages | 7 |
| Journal | Molecular Immunology |
| Volume | 46 |
| Issue number | 15 |
| DOIs | |
| State | Published - Sep 2009 |
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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 journal › Article
}
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 -