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dc.contributor.authorMeiring, Hugo D
dc.contributor.authorSoethout, Ernst C
dc.contributor.authorPoelen, Martien C M
dc.contributor.authorMooibroek, Dennis
dc.contributor.authorHoogerbrugge, Ronald
dc.contributor.authorTimmermans, Hans
dc.contributor.authorBoog, Claire J
dc.contributor.authorHeck, Albert J R
dc.contributor.authorJong, Ad P J M de
dc.contributor.authorEls, Cécile A C M van
dc.date.accessioned2007-02-14T15:05:51Z
dc.date.available2007-02-14T15:05:51Z
dc.date.issued2006-05-01
dc.identifier.citationMol. Cell Proteomics 2006, 5(5):902-13en
dc.identifier.issn1535-9476
dc.identifier.pmid16432254
dc.identifier.doi10.1074/mcp.T500014-MCP200
dc.identifier.urihttp://hdl.handle.net/10029/8396
dc.description.abstractIdentification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.
dc.format.extent1102412 bytes
dc.format.mimetypeapplication/pdf
dc.language.isoenen
dc.titleStable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.en
dc.typeArticleen
dc.format.digYES
refterms.dateFOA2018-12-18T15:35:01Z
html.description.abstractIdentification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.


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