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Webbased Archive of RIVM Publications > Articles and other publications by RIVM employees > Vaccines and Vaccination > Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.


Please use this identifier to cite or link to this item: http://hdl.handle.net/10029/8396
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Title: Stable isotope tagging of epitopes: a highly selective strategy for the identification of major histocompatibility complex class I-associated peptides induced upon viral infection.
Authors: Meiring, Hugo D
Soethout, Ernst C
Poelen, Martien C M
Mooibroek, Dennis
Hoogerbrugge, Ronald
Timmermans, Hans
Boog, Claire J
Heck, Albert J R
Jong, Ad P J M de
Els, Cécile A C M van
Citation: Mol. Cell Proteomics 2006, 5(5):902-13
Issue Date: 1-May-2006
URI: http://hdl.handle.net/10029/8396
DOI: 10.1074/mcp.T500014-MCP200
PubMed ID: 16432254
Abstract: Identification 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.
Type: Article
Language: en
ISSN: 1535-9476
Appears in Collections:  Vaccines and Vaccination

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