UNIT 16.3 Targeted Identification of Infection-Related HLA Class I–Presented Epitopes by Stable Isotope Tagging of Epitopes (SITE)
Published Online: 1 MAY 2007
Copyright © 2007 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Meiring, H., Soethout, E., de Jong, A. and van Els, C. 2007. Targeted Identification of Infection-Related HLA Class I–Presented Epitopes by Stable Isotope Tagging of Epitopes (SITE). Current Protocols in Immunology. 77:16.3:16.3.1–16.3.20.
- Published Online: 1 MAY 2007
- Published Print: MAY 2007
Identification of peptides presented in human leukocyte antigen (HLA) class I molecules after viral infection is of strategic importance for immunology and vaccine development. A powerful strategy aimed at the rapid, unambiguous identification of naturally processed HLA class I–associated peptides, which are induced by viral infection, is presented here. 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 for the human leukocyte antigen allele of interest. Subsequently, these cells are mixed with an equal number of noninfected cells, which are cultured in normal medium. Finally, peptides are acid-eluted from immunoprecipitated HLA molecules and subjected to two-dimensional nanoscale liquid chromatography–mass spectrometry analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules.
- HLA class I;
- mass spectrometry;
- CD8+ T cell