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 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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We would like to thank Lynne Yakes for help in the preparation of this manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB510) and the European Union (QLQ2-CT-1999-00713).
The authors declare no competing financial interests.
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Lemmel, C., Weik, S., Eberle, U. et al. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling. Nat Biotechnol 22, 450–454 (2004) doi:10.1038/nbt947
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