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.
Highlights
Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development
The adaptive immune response to virus infections is characterized by the generation of CD8ϩ cytotoxic T lymphocytes (CTLs),1 which recognize peptides presented by MHC class I molecules of infected cells
stable isotope tagging of epitopes (SITE): Stable Isotope Tagging of Epitopes though healthy children clear these infections in 7–21 days, children with disorders in the CTL response may suffer from complications after measles virus (MV) [13, 14] or respiratory syncytial virus (RSV) infection [15]
Summary
Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. We successfully used the mass spectrometric method to identify novel MHC class I-restricted viral epitopes by subtractive analysis of peptide pools isolated from virus-infected and control cells [9].
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.