Glycoprotein Enrichment Through Lectin Affinity Techniques

  • Yehia Mechref
  • Milan Madera
  • Milos V Novotny
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)


Posttranslational modifications (PTM) of proteins are among the key biological regulators of function, activity, localization, and interaction. The fact that no more than 30,000–50,000 proteins are encoded by the human genome underlines the importance of posttranslational modifications in modulating the activities and functions of proteins in health and disease. With approximately 50% of all proteins now considered to be glycosylated, its physiological importance in mammalian systems is imperative. Aberrant glycosylation has now been recognized as an attribute of many mammalian diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular conditions, and cancer. As many potential disease biomarkers may be glycoproteins present in only minute quantities in tissue extracts and physiological fluids, glycoprotein isolation and enrichment may be critical in a search for such biomarkers. For decades, efforts have been focused on the development of glycoprotein enrichment from complex biological samples. Logically, the great majority of these enrichment methodologies rely on the use of immobilized lectins, which permit selective enrichment of the pools of glycoproteins for proteomic/glycomic studies. In this chapter, lectin affinity chromatography in different formats are described, including tubes; packed columns, and microfluidic channels.

Key Words

Agarose lectin material glycoprotein enrichment human blood serum glycoproteins lectin affinity chromatography silica-based lectin material 



This work was supported by Grant No. GM24349 from the National Institute of General Medical Sciences, U.S. Department of Health and Human Services and a center grant from the Indiana 21st Century Research and Technology Fund. This work was also supported by the National Institute of Health through the National Center for Research Resources (NCRR) by grant number RR018942 for the National Center for Glycomics and Glycoproteomics.


  1. 1.
    Kellner, R., Lottspeich, F., and Meyer, H. E. (1999) Microcharacterization of Proteins, Wiley-Vch, Weinheim.Google Scholar
  2. 2.
    Walker, J. M. (Ed.) (1996) The Protein Protocols Handbook, Humana Press, Humana Press, Totowa.Google Scholar
  3. 3.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–85.CrossRefPubMedGoogle Scholar
  4. 4.
    Cummings, R. D. (1994) Use of Lectins in Analysis of Glycoconjugates. Methods Enzymol. 230, 66–86.CrossRefPubMedGoogle Scholar
  5. 5.
    Gravel, P., Walzer, C., Aubry, C., Balant, L. P., Yersin, B., Hochstrasser, D. F., and Guimon, J. (1996) New alterations of serum glycoproteins in alcoholic and cirrhotic patients revealed by high resolution two-dimensional gel . Biochem. Biophys. Res. Commun. 220, 78–85.CrossRefGoogle Scholar
  6. 6.
    Carlsson, J., Janson, J.-C., and Sparrman, M. (1989) Affinity chromatography, in Protein Purification (Janson, J.-C., and Ryd’en, L., eds.), VCH Publishers, Inc., Uppsala, pp. 275–329.Google Scholar
  7. 7.
    Lotan, R., Beattie, G., Hubbell, W., and Nicolson, G. L. (1977) Activities of lectins and their immobilized derivatives in detergent solutions – implications on use of lectin affinity chromatography for purification of membrane glycoproteins. Biochemistry 16, 1787–94.CrossRefPubMedGoogle Scholar
  8. 8.
    Kinzel, V., Kubler, D., Richards, J., and Stohr, M. (1976) Lens Culinaris Lectin Immobilized on Sepharose – Binding and Sugar-Specific Release of Intact Tissue-Culture Cells. Science 192, 487–89.CrossRefPubMedGoogle Scholar
  9. 9.
    Mechref, Y., Ma, W., Hao, G., and Novotny, M. V. (1999) N-Linked oligosaccharides of vomeromodulin, a putative pheromone transporter in rat. Biochem. Biophys. Res. Commun. 255, 451–55.CrossRefPubMedGoogle Scholar
  10. 10.
    Mechref, Y., Zidek, L., Ma, W., and Novotny, M. V. (2000) Glycosylated major urinary protein of the mouse: characterization of its N-linked oligosaccharides. Glycobiology 10, 231–35.CrossRefPubMedGoogle Scholar
  11. 11.
    Nawarak, J., Phutrakul, S., and Chen, S.-T. (2004) Analysis of lectin-bound glycoproteins in snake venom from the elapidae and viperidae families. J. Proteome Res. 3, 383–92.CrossRefPubMedGoogle Scholar
  12. 12.
    Larsson, P.-O. (1984) High-performance liquid affinity chromatography. Methods Enzymol. 104, 212–23.CrossRefPubMedGoogle Scholar
  13. 13.
    Voyksner, R. D., Chen, D. C., and Swaisgood, H. E. (1990) Optimization of immobilized enzyme hydrolysis combined with high-performance liquid-chromatography thermospray mass-spectrometry for the determination of neuropeptides. Anal. Biochem. 188, 72–81.CrossRefPubMedGoogle Scholar
  14. 14.
    Borrebaeck, C. A. K., Soares, J., and Mattiasson, B. (1984) Fractionation of glycoproteins according to lectin affinity and molecular-size using a high-performance liquid-chromatography system with sequentially coupled columns. J. Chromatogr. 284, 187–92.CrossRefGoogle Scholar
  15. 15.
    Green, E. D., Brodbeck, R. M., and Baenziger, J. U. (1987) Lectin affinity high-performance liquid-chromatography – interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin-A, datura-stramonium agglutinin, and vicia-villosa agglutinin. Anal. Biochem. 167, 62–75.CrossRefPubMedGoogle Scholar
  16. 16.
    Madera, M., Mechref, Y., Klouckova, I., and Novotny, M. V. (2006) Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry. J. Proteome Res., 5, 2348–63.CrossRefPubMedGoogle Scholar
  17. 17.
    Palm, A., and Novotny, M. V. (1997) Macroporous Polyacrylamide/Poly(ethylene glycol) Matrixes as Stationary Phases in Capillary Electrochromatography Anal. Chem. 69, 4499–507.CrossRefGoogle Scholar
  18. 18.
    Palm, A. K., and Novotny, M. V. (2004) Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry. Rapid Commun. Mass Spectrom. 18, 1374–82.CrossRefPubMedGoogle Scholar
  19. 19.
    Palm, A. K., and Novotny, M. V. (2005) A monolithic PNGase F enzyme microreactor enabling glycan mass mapping of glycoproteins by mass spectrometry. Rapid Commun. Mass Spectrom. 19, 1730–38.CrossRefPubMedGoogle Scholar
  20. 20.
    Williams, S. L., Eccleston, M. E., and Slater, N. K. H. (2005) Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: Towards a rapid single-step purification process. Biotechnol. Bioeng. 89, 783–87.CrossRefPubMedGoogle Scholar
  21. 21.
    Que, A. H., Mechref, Y., Huang, Y., Taraszka, J. A., Clemmer, D. E., and Novotny, M. V. (2003) Coupling capillary electrochromatography with electrospray Fourier transform mass spectrometry for characterizing complex oligosaccharide pools. Anal. Chem. 75, 1684–90.CrossRefPubMedGoogle Scholar
  22. 22.
    Guo, L., Eisenman, J. R., Mahimkar, R. M., Peschon, J. J., Paxton, R. J., Black, R. A., and Johnson, R. S. (2002) A proteomic approach for the identification of cell-surface proteins shed by metalloproteases. Mol. Cell. Proteomics 1, 30–36.CrossRefPubMedGoogle Scholar
  23. 23.
    Josic, D., and Buchacher, A. (2001) Application of monoliths as supports for affinity chromatography and fast enzymatic conversion. J. Biochem. Bioph. Methods 49, 153–74.CrossRefGoogle Scholar
  24. 24.
    Josic, D., Buchacher, A., and Jungbauer, A. (2001) Monoliths as stationary phases for setion of proteins and polynucleotides and enzymatic conversion. J. Chromatogr. B 752, 191–205.CrossRefGoogle Scholar
  25. 25.
    Svec, F. (2001) Capillary column technology: continuous polymer monoliths, in Capillary Electrochromatography (Deyl, Z., and Svec, F., Eds.), Elsevier, Amesterdam, pp. 183–240.Google Scholar
  26. 26.
    Svec, F., Peters, E. C., Sykora, D., and Frechet, J. M. J. (2000) Design of the monolithic polymers used in capillary electrochromatography columns. J. Chromatogr. A 887, 3–29.CrossRefPubMedGoogle Scholar
  27. 27.
    Pan, Z., Zou, H., Mo, W., Huang, X., and Wu, R. (2002) Protein A immobilized monolithic capillary column for affinity chromatography. Anal. Chim. Acta 466, 141–50.Google Scholar
  28. 28.
    Bedair, M., and Rassi, Z. E. (2004) Affinity chromatography with monolithic capillary columns I. Polymethacrylate monoliths with immobilized mannan for thye setion of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography. J. Chromatogr. A 1044, 177–86.CrossRefGoogle Scholar
  29. 29.
    Bedair, M., and Rassi, Z. E. (2005) Affinity chromatography with monolithic capillary columns: II. Polymethacrylate monoliths with immobilized lectins for the setion of glycoconjugates by nano-liquid affinity chromatography. J. Chromatogr. A 1079, 236–45.CrossRefPubMedGoogle Scholar
  30. 30.
    Mao, X., Luo, Y., Dai, Z., Wang, K., Du, Y., and Lin, B. (2004) Integrated lectin affinity microfluidic chip for glycoform setion. Anal. Chem. 76, 6941–47.CrossRefPubMedGoogle Scholar
  31. 31.
    Madera, M., Mechref, Y., and Novotny, M. V. (2005) Combining lectin microcolumns with high-resolution separation techniques for enrichment of glycoproteins and glycopeptides. Anal. Chem. 77, 4081–90.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Yehia Mechref
    • 1
  • Milan Madera
    • 1
  • Milos V Novotny
    • 1
  1. 1.Indiana UniversityBloomingtonIndiana

Personalised recommendations