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.

  • Article
  • Published:

Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling

Nature Methods volume 2, pages 851–856 (2005)Cite this article

Abstract

Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, Kd > 10−6 M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: The power of the evanescent-field fluorescence-assisted lectin microarray platform.
Figure 2: Quantitative analysis of lectin-glycoprotein interaction.
Figure 3: Challenge of glycan profiling with a 39-lectin microarray platform.
Figure 4: Monitoring of the sequential digestion of glycan with lectin microarray.

Similar content being viewed by others

References

  1. Apweiler, R., Hermjakob, H. & Sharon, N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta 1473, 4–8 (1999).

    Article  CAS  Google Scholar 

  2. Varki, A., Cummings, R.D., Esko, J., Freeze, H. & Hart, G. Essentials of glycobiology. (Cold Spring Harbor Laboratory, New York, 1999).

  3. Feizi, T. Progress in deciphering the information content of the 'glycome'—a crescendo in the closing years of the millennium. Glycoconj. J. 17, 553–565 (2000).

    Article  CAS  Google Scholar 

  4. Hirabayashi, J. & Kasai, K. Glycomics, coming of age! Trends Glycosci. Glycotechnol. 12, 1–5 (2000).

    Article  Google Scholar 

  5. Hirabayashi, J. Lectin-based structural glycomics: glycoproteomics and glycan profiling. Glycoconj. J. 21, 35–40 (2004).

    Article  Google Scholar 

  6. Hirabayashi, J., Kaji, H., Isobe, T. & Kasai, K. Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans. J. Biochem. (Tokyo) 132, 103–114 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Bochner, B.S. et al. Glycan array screening reveals a candidate ligand for Siglec-8. J. Biol. Chem. 280, 4307–4312 (2005).

    Article  CAS  Google Scholar 

  9. Kasai, K. & Ishii, S. Quantitative analysis of affinity chromatography of trypsin. A new technique for investigation of protein-ligand interaction. J. Biochem. (Tokyo) 77, 261–264 (1975).

    CAS  Google Scholar 

  10. Feizi, T., Fazio, F., Chai, W. & Wong, C.H. Carbohydrate microarrays - a new set of technologies at the frontiers of glycomics. Curr. Opin. Struct. Biol. 13, 637–645 (2003).

    Article  CAS  Google Scholar 

  11. Wang, D., Liu, S., Trummer, B.J., Deng, C. & Wang, A. Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol. 20, 275–281 (2002).

    Article  CAS  Google Scholar 

  12. Fukui, S., Feizi, T., Galustian, C., Lawson, A.M. & Chai, W. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 20, 1011–1017 (2002).

    Article  CAS  Google Scholar 

  13. Fazio, F., Bryan, M.C., Blixt, O., Paulson, J.C. & Wong, C.H. Synthesis of sugar arrays in microtiter plate. J. Am. Chem. Soc. 124, 14397–14402 (2002).

    Article  CAS  Google Scholar 

  14. 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  Google Scholar 

  15. Ratner, D.M. et al. Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides. ChemBioChem 5, 379–383 (2004).

    Article  CAS  Google Scholar 

  16. Ratner, D.M., Adams, E.W., Disney, M.D. & Seeberger, P.H. Tools for glycomics: mapping interactions of carbohydrates in biological systems. ChemBioChem 5, 1375–1383 (2004).

    Article  CAS  Google Scholar 

  17. Stimpson, D.I. et al. Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides. Proc. Natl. Acad. Sci. USA 92, 6379–6383 (1995).

    Article  CAS  Google Scholar 

  18. Lehr, H.P., Reimann, M., Brandenburg, A., Sulz, G. & Klapproth, H. Real-time detection of nucleic acid interactions by total internal reflection fluorescence. Anal. Chem. 75, 2414–2420 (2003).

    Article  CAS  Google Scholar 

  19. Angeloni, S. et al. Glycoprofiling with micro-arrays of glycoconjugates and lectins. Glycobiology 15, 31–41 (2005).

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Shinohara, Y. et al. Kinetic measurement of the interaction between an oligosaccharide and lectins by a biosensor on surface plasmon resonance. Eur. J. Biochem. 223, 189–194 (1994).

    Article  CAS  Google Scholar 

  22. Mizuno, M., Noguchi, M., Imai, M., Motoyoshi, T. & Inazu, T. Interaction assay of oligosaccharide with lectins using glycosylasparagine. Bioorg. Med. Chem. Lett. 14, 485–490 (2004).

    Article  CAS  Google Scholar 

  23. Yang, L., Tang, Q., Harrata, A.K. & Lee, C.S. Capillary isoelectric focusing-electrospray ionization mass spectrometry for transferrin glycoforms analysis. Anal. Biochem. 243, 140–149 (1996).

    Article  CAS  Google Scholar 

  24. Knibbs, R.N., Perini, F. & Goldstein, I.J. Structure of the major concanavalin A reactive oligosaccharides of the extracellular matrix component laminin. Biochemistry 28, 6379–6392 (1989).

    Article  CAS  Google Scholar 

  25. Wu, A.M., Wu, J.H., Herp, A. & Liu, J-H. Effect of polyvalencies of glycotopes on the binding of a lectin from the edible mushroom, Agaricus bisporus. Biochem. J. 371, 311–320 (2003).

    Article  CAS  Google Scholar 

  26. Wuhrer, M., Hokke, C.H. & Deelder, A.M. Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation. Rapid Commun. Mass Spectrom. 18, 1741–1748 (2004).

    Article  CAS  Google Scholar 

  27. Wimmerova, M., Mitchell, E., Sanchez, J.F., Gautier, C. & Imberty, A. Crystal structure of fungal lectin: six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin. J. Biol. Chem. 278, 27059–27067 (2003).

    Article  CAS  Google Scholar 

  28. Zhang, H., Li, X-J., Martin, D.B. & Aebersold, R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666 (2003).

    Article  CAS  Google Scholar 

  29. Kaji, H. et al. Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins. Nat. Biotechnol. 21, 667–672 (2003).

    Article  CAS  Google Scholar 

  30. Zhu, H. et al. Analysis of yeast protein kinases using protein chips. Nat. Genet. 26, 283–289 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Nakamura, AIST, and K. Kasai, Teikyo University, for critical discussion about the lectin-carbohydrate interaction. We also thank M. Mizuno, the Noguchi Institute, and K. Hayama, AIST, for helpful advice about preparation of TAMRA-labeled oligosaccharide and glycopeptide mixture derived from ASF, respectively. This work was supported in part by New Energy and Industrial Technology Development Organization (NEDO) in Japan.

Author information

Author notes

  1. Atsushi Kuno and Noboru Uchiyama: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2,

    Atsushi Kuno, Noboru Uchiyama, Shiori Koseki-Kuno & Jun Hirabayashi

  2. Tsukuba, 305-8568, Ibaraki, Japan

    Atsushi Kuno, Noboru Uchiyama, Shiori Koseki-Kuno & Jun Hirabayashi

  3. Moritex Co., Yokohama, 225-0012, Kanagawa, Japan

    Youji Ebe & Masao Yamada

  4. Nippon Laser & Electronics Lab., Nagoya, 456-0032, Aichi, Japan

    Seigo Takashima & Masao Yamada

Authors
  1. Atsushi Kuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Noboru Uchiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shiori Koseki-Kuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Youji Ebe
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Seigo Takashima
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masao Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jun Hirabayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jun Hirabayashi.

Ethics declarations

Competing interests

Y.E. is employed by Moritex Co. and S.T. is employed by Nippon Laser & Electronics Lab. M.Y. is employed by both of these companies.

Supplementary information

Supplementary Fig. 1

Principle of the evanescent-field fluorescence-detection system and cutting model illustration of an optical scheme for the detection system. (PDF 224 kb)

Supplementary Fig. 2

Glycoproteins used in this study. (PDF 92 kb)

Supplementary Fig. 3

Determination of the Kd value of every lectin-glycan interaction. (PDF 21 kb)

Supplementary Fig. 4

Detection of lectin-glycopeptide interaction with lectin microarray. (PDF 257 kb)

Supplementary Table 1

Abbreviations for 39 lectins and their sugar binding specificities. (PDF 43 kb)

Supplementary Table 2

Standardization of signal intensity with hybrid array. (PDF 13 kb)

Supplementary Methods (PDF 27 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kuno, A., Uchiyama, N., Koseki-Kuno, S. et al. Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling. Nat Methods 2, 851–856 (2005). https://doi.org/10.1038/nmeth803

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth803

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