Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling

Article metrics


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Strategy for differential quantification of MHC-eluted peptides.
Figure 2: MS spectra before and after acetylation.
Figure 3: Differential quantification of HLA ligands derived from two different sources.
Figure 4: Recoveries of different peptide derivatives.


  1. 1

    Riordan, J.F. Acetylation. Methods Enzymol. 11, 565–570 (1967).

  2. 2

    Beardsley, R.L. & Reilly, J.P. Optimization of guanidination procedures for MALDI mass mapping. Anal. Chem. 74, 1884–1890 (2002).

  3. 3

    Munchbach, M., Quadroni, M., Miotto, G. & James, P. Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety. Anal. Chem. 72, 4047–4057 (2000).

  4. 4

    Ji, J. et al. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B Biomed. Sci. Appl. 745, 197–210 (2000).

  5. 5

    Wang, Q. et al. Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer. Gene 263, 205–209 (2001).

  6. 6

    Ougolkov, A.V., Yamashita, K., Mai, M. & Minamoto, T. Oncogenic beta-catenin and MMP-7 (matrilysin) cosegregate in late-stage clinical colon cancer. Gastroenterology 122, 60–71 (2002).

  7. 7

    Bienz, M. & Clevers, H. Linking colorectal cancer to Wnt signaling. Cell 103, 311–320 (2000).

  8. 8

    Bienz, M. & Clevers, H. Armadillo/beta-catenin signals in the nucleus—proof beyond a reasonable doubt? Nat. Cell Biol. 5, 179–182 (2003).

  9. 9

    Zechner, D. et al. Beta-catenin signals regulate cell growth and the balance between progenitor cell expansion and differentiation in the nervous system. Dev. Biol. 258, 406–418 (2003).

  10. 10

    Shih, I.M., Yu, J., He, T.C., Vogelstein, B. & Kinzler, K.W. The beta-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression. Cancer Res. 60, 1671–1676 (2000).

  11. 11

    Rammensee, H., Bachmann, J., Emmerich, N.P., Bachor, O.A. & Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50, 213–219 (1999).

  12. 12

    Robbins, P.F. et al. A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J. Exp. Med. 183, 1185–1192 (1996).

  13. 13

    Kimmel, J.R. Analysis of homoarginine. Methods Enzymol. 11, 584–589 (1967).

  14. 14

    Trask, D.K. et al. Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells. Proc. Natl. Acad. Sci. USA 87, 2319–2323 (1990).

  15. 15

    Fossar, N., Chaouche, M., Prochasson, P., Rousset, M. & Brison, O. Deregulated expression of the keratin 18 gene in human colon carcinoma cells. Somat. Cell Mol. Genet. 25, 223–235 (1999).

  16. 16

    Chu, Y.W., Seftor, E.A., Romer, L.H. & Hendrix, M.J. Experimental coexpression of vimentin and keratin intermediate filaments in human melanoma cells augments motility. Am. J. Pathol. 148, 63–69 (1996).

  17. 17

    Weinschenk, T. et al. Integrated functional genomics approach for the design of patient-individual antitumor vaccines. Cancer Res. 62, 5818–5827 (2002).

  18. 18

    Hunt, D.F. et al. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263 (1992).

  19. 19

    Luckey, C.J. et al. Differences in the expression of human class I MHC alleles and their associated peptides in the presence of proteasome inhibitors. J. Immunol. 167, 1212–1221 (2001).

  20. 20

    Herberts, C.A. et al. Autoreactivity against induced or upregulated abundant self-peptides in HLA-A*0201 following measles virus infection. Hum. Immunol. 64, 44–55 (2003).

  21. 21

    Macdonald, W.A. et al. A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition. J. Exp. Med. 198, 679–691 (2003).

  22. 22

    Zhang, R., Sioma, C.S., Wang, S. & Regnier, F.E. Fractionation of isotopically labeled peptides in quantitative proteomics. Anal. Chem. 73, 5142–5149 (2001).

  23. 23

    Hansen, K.C. et al. Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography. Mol. Cell Proteomics 2, 299–314 (2003).

  24. 24

    Schirle, M. et al. Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach. Eur. J. Immunol. 30, 2216–2225 (2000).

  25. 25

    Falk, K., Rotzschke, O., Stevanovic, S., Jung, G. & Rammensee, H.G. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351, 290–296 (1991).

  26. 26

    Seeger, F.H. et al. The HLA-A*6601 peptide motif: prediction by pocket structure and verification by peptide analysis. Immunogenetics 49, 571–576 (1999).

  27. 27

    Morris, H.R. et al. High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 10, 889–896 (1996).

Download references


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).

Author information

Correspondence to Stefan Stevanović.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading