Nature Biotechnology
22, 450 - 454 (2004)
Published online: 7 March 2004; | doi:10.1038/nbt947
Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labelingClaudia Lemmel1, Steffen Weik2, Ute Eberle1, Jörn Dengjel1, Thomas Kratt3, Horst-Dieter Becker3, Hans-Georg Rammensee1
& Stefan Stevanovi 11
Department of Immunology, Institute for Cell Biology, University of Tübingen, D-72076 Tübingen, Germany. 2
Institute for Organic Chemistry, University of Tübingen, D-72076 Tübingen, Germany. 3
Department of General Surgery, University of Tübingen, D-72076 Tübingen, Germany.
Correspondence should be addressed to Stefan Stevanovi stefan.stevanovic@uni-tuebingen.deCurrently, 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 acetylation1 and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination2 and nicotinylation3, 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.
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