Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays

Nature Methods volume 4, pages 437–444 (2007)Cite this article

Abstract

Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Detection of glycans on antibody arrays.
Figure 2: Optimization and characterization of chemical derivatization.
Figure 3: The effect of derivatization on antibody affinities.
Figure 4: Lectin binding profiles of native and enzyme-digested protein.
Figure 5: Profiling protein and glycan variation in cancer and control sera.

References

  1. Dube, D.H. & Bertozzi, C.R. Glycans in cancer and inflammation–potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 4, 477–488 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Dennis, J.W., Granovsky, M. & Warren, C.E. Glycoprotein glycosylation and cancer progression. Biochim. Biophys. Acta 1473, 21–34 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Fuster, M.M. & Esko, J.D. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat. Rev. Cancer 5, 526–542 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. van Dijk, W., Havenaar, E.C. & Brinkman-van der Linden, E.C. Alpha 1-acid glycoprotein (orosomucoid): pathophysiological changes in glycosylation in relation to its function. Glycoconj. J. 12, 227–233 (1995).

    Article  CAS  PubMed  Google Scholar 

  5. Okuyama, N. et al. Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation. Int. J. Cancer 118, 2803–2808 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Thompson, S., Stappenbeck, R. & Turner, G.A. A multiwell lectin-binding assay using lotus tetragonolobus for measuring different glycosylated forms of haptoglobin. Clin. Chim. Acta 180, 277–284 (1989).

    Article  CAS  PubMed  Google Scholar 

  7. Kuno, A. et al. Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling. Nat. Methods 2, 851–856 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Pilobello, K.T., Krishnamoorthy, L., Slawek, D. & Mahal, L.K. Development of a lectin microarray for the rapid analysis of protein glycopatterns. ChemBioChem 6, 985–989 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Zheng, T., Peelen, D. & Smith, L.M. Lectin arrays for profiling cell surface carbohydrate expression. J. Am. Chem. Soc. 127, 9982–9983 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Shimizu, K. et al. Comparison of carbohydrate structures of serum alpha-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis. Clin. Chim. Acta 254, 23–40 (1996).

    Article  CAS  PubMed  Google Scholar 

  11. Thompson, S., Cantwell, B.M., Cornell, C. & Turner, G.A. Abnormally-fucosylated haptoglobin: a cancer marker for tumour burden but not gross liver metastasis. Br. J. Cancer 64, 386–390 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Thompson, S., Guthrie, D. & Turner, G.A. Fucosylated forms of alpha-1-antitrypsin that predict unresponsiveness to chemotherapy in ovarian cancer. Br. J. Cancer 58, 589–593 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kjeldsen, T. et al. Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2-6 alpha-N-acetylgalactosaminyl (sialosyl-Tn) epitope. Cancer Res. 48, 2214–2220 (1988).

    CAS  PubMed  Google Scholar 

  14. Hanisch, F.G., Hanski, C. & Hasegawa, A. Sialyl Lewis(x) antigen as defined by monoclonal antibody AM-3 is a marker of dysplasia in the colonic adenoma-carcinoma sequence. Cancer Res. 52, 3138–3144 (1992).

    CAS  PubMed  Google Scholar 

  15. Burchell, J. et al. Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res. 47, 5476–5482 (1987).

    CAS  PubMed  Google Scholar 

  16. Hirabayashi, J. et al. Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim. Biophys. Acta 1572, 232–254 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Van Damme, E.J.M., Peumans, W.J., Pusztai, A. & Bardocz, S. Handbook of Plant Lectins: Properties and Biomedical Applications (John Wiley & Sons, Chichester, 1998).

    Google Scholar 

  18. Manimala, J.C., Roach, T.A., Li, Z. & Gildersleeve, J.C. High-throughput carbohydrate microarray analysis of 24 lectins. Angew. Chem. Int. Edn Engl. 45, 3607–3610 (2006).

    Article  CAS  Google Scholar 

  19. Roberts, G.P. Histochemical detection of sialic acid residues using periodate oxidation. Histochem. J. 9, 97–102 (1977).

    Article  CAS  PubMed  Google Scholar 

  20. Yamashita, K., Koide, N., Endo, T., Iwaki, Y. & Kobata, A. Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma. J. Biol. Chem. 264, 2415–2423 (1989).

    CAS  PubMed  Google Scholar 

  21. Reis, C.A., David, L., Seixas, M., Burchell, J. & Sobrinho-Simoes, M. Expression of fully and under-glycosylated forms of MUC1 mucin in gastric carcinoma. Int. J. Cancer 79, 402–410 (1998).

    Article  CAS  PubMed  Google Scholar 

  22. Lloyd, K.O., Burchell, J., Kudryashov, V., Yin, B.W. & Taylor-Papadimitriou, J. Comparison of O-linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines. Demonstration of simpler and fewer glycan chains in tumor cells. J. Biol. Chem. 271, 33325–33334 (1996).

    Article  CAS  PubMed  Google Scholar 

  23. Ho, J.J., Siddiki, B. & Kim, Y.S. Association of sialyl-Lewis(a) and sialyl-Lewis(x) with MUC-1 apomucin in a pancreatic cancer cell line. Cancer Res. 55, 3659–3663 (1995).

    CAS  PubMed  Google Scholar 

  24. Kalthoff, H., Kreiker, C., Schmiegel, W.H., Greten, H. & Thiele, H.G. Characterization of CA 19–9 bearing mucins as physiological exocrine pancreatic secretion products. Cancer Res. 46, 3605–3607 (1986).

    CAS  PubMed  Google Scholar 

  25. von Ritter, C. et al. Biliary mucin secreted by cultured human gallbladder epithelial cells carries the epitope of CA 19–9. Anticancer Res. 17, 2931–2934 (1997).

    CAS  PubMed  Google Scholar 

  26. Lucka, L. et al. Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes. Glycobiology 15, 87–100 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Stocks, S.C., Albrechtsen, M. & Kerr, M.A. Expression of the CD15 differentiation antigen (3-fucosyl-N-acetyl-lactosamine, LeX) on putative neutrophil adhesion molecules CR3 and NCA-160. Biochem. J. 268, 275–280 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Stocks, S.C. & Kerr, M.A. Neutrophil NCA-160 (CD66) is the major protein carrier of selectin binding carbohydrate groups LewisX and sialyl lewisX. Biochem. Biophys. Res. Commun. 195, 478–483 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Blixt, O. et al. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. USA 101, 17033–17038 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Manimala, J.C., Li, Z., Jain, A., VedBrat, S. & Gildersleeve, J.C. Carbohydrate array analysis of anti-Tn antibodies and lectins reveals unexpected specificities: implications for diagnostic and vaccine development. ChemBioChem 6, 2229–2241 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Mistry for assistance with the experiments and other members of the Laboratory of Cancer Immunodiagnostics at the Van Andel Research Institute for helpful interactions. We thank A. Rai (Memorial Sloan Kettering Cancer Center) and I. Goldstein (University of Michigan) for valuable input. We gratefully acknowledge the Van Andel Research Institute for support of this work.

Author information

Author notes

  1. Tom LaRoche & Darren Hamelinck

    Present address: Present addresses: Wayne State University Medical School, 540 E. Canfield, Detroit, MI 48201, USA (T.L.), McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4L8 (D.H.).,

Authors and Affiliations

  1. Van Andel Research Institute, 333 Bostwick, Grand Rapids, 49503, Michigan, USA

    Songming Chen, Tom LaRoche, Darren Hamelinck, Derek Bergsma & Brian B Haab

  2. University of Michigan Medical Center, 1500 E. Hospital Drive, Ann Arbor, 48109, Michigan, USA

    Dean Brenner & Diane Simeone

  3. Evanston Northwestern Healthcare, 2100 Pfingstein Rd, Glenview, 60025, Illinois, USA

    Randall E Brand

Authors
  1. Songming Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tom LaRoche
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Darren Hamelinck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Derek Bergsma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dean Brenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Diane Simeone
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Randall E Brand
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Brian B Haab
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian B Haab.

Ethics declarations

Competing interests

B.B.H. and S.C. are inventors on a patent application covering the technology described in this article. This technology has been licensed for commercialization by GenTel Biosciences (Madison, Wisconsin, USA).

Supplementary information

Supplementary Fig. 1

Chemical derivatization of antibodies to block lectin binding. (PDF 507 kb)

Supplementary Fig. 2

Optimization of antibody derivatization. (PDF 4163 kb)

Supplementary Fig. 3

Optimization of NaIO4 oxidation. (PDF 509 kb)

Supplementary Fig. 4

Effects of NaIO4 concentration on antibody retainment and background signals. (PDF 532 kb)

Supplementary Table 1

Lectins used in the experiments and their specificities. (PDF 133 kb)

Supplementary Table 2

Measured EC50s of 24 lectins for the glycans of spotted antibodies. (PDF 531 kb)

Supplementary Table 3

Antibodies and proteins used in the experiments. (PDF 21 kb)

Supplementary Methods (PDF 21 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chen, S., LaRoche, T., Hamelinck, D. et al. Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays. Nat Methods 4, 437–444 (2007). https://doi.org/10.1038/nmeth1035

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth1035

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing