Targeted identification of infection-related HLA class I-presented epitopes by stable isotope tagging of epitopes (SITE).
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.
Meiring
H D
HD
Netherlands Vaccine Institute, Bilthoven, The Netherlands.
Soethout
E C
EC
Netherlands Vaccine Institute, Bilthoven, The Netherlands.
de Jong
A P J M
APJM
Netherlands Vaccine Institute, Bilthoven, The Netherlands.
van Els
C A C M
CACM
Netherlands Vaccine Institute, Bilthoven, The Netherlands.
D017951:Antigen Presentation; D016010:Binomial Distribution; D002852:Chromatography, Ion Exchange / Q000379:methods; D006680:HLA Antigens / Q000276:immunology / Q000302:isolation & purification*; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D007158:Immunologic Techniques; D007553:Isotope Labeling; D011499:Protein Processing, Post-Translational; D012815:Signal Processing, Computer-Assisted; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014777:Virus Diseases / Q000276:immunology
D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D016056:Immunodominant Epitopes