Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.
Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.
Claudia
C
Lemmel
Steffen
S
Weik
Ute
U
Eberle
Jörn
J
Dengjel
Thomas
T
Kratt
Horst-Dieter
HD
Becker
Hans-Georg
HG
Rammensee
Stefan
S
Stevanovic
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D018076:DNA, Complementary / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007554:Isotopes / Q000737:chemistry*; D008024:Ligands; D008285:Major Histocompatibility Complex; D013058:Mass Spectrometry / Q000379:methods*; D009538:Nicotine / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014162:Transfection
D018076:DNA, Complementary; D007554:Isotopes; D008024:Ligands; D010455:Peptides; D009538:Nicotine; D019791:Guanidine