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Polyacrylamide Gel Electrophoresis of Fluorophore-Labeled Carbohydrates from Glycoproteins

  • Brian K. Brandley
  • John C. Klock
  • Christopher M. Starr
Protocol
  • 99 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Prior to 1980, most methods for analysis of glycoprotein carbohydrates utilized column, thin-layer, and paper chromatography, gas chromatography, mass spectroscopy, and rarely nuclear magnetic resonance spectroscopy. These methods required relatively large amounts of materials (micromoles), specialized training and experience, and in some cases, significant capital equipment outlays. Because of these restrictions, convenient carbohydrate analysis on small samples was not available to most biologists. Recently, improvements in chromatographic methods, labeling methods for carbohydrates, carbohydrate-specific enzymes, and higher resolution electrophoresis methods have allowed carbohydrate analysis to be done on nanomolar amounts of material. Because of these improvements, today’s biologist now has an improved ability to evaluate the role of carbohydrates in their research and development work.

Keywords

Sodium Dodecyl Sulfate Sodium Cyanoborohydride Electrophoresis Tank Bovine Submaxillary Mucin Fluorophore Label 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Williams, G. R. and Jackson, P. (1992) Analysis of carbohydrates. U.S. Patent 5, 508.Google Scholar
  2. 2.
    Jackson, P. (1990) The use of polyacrylamide-gel electrophoresis for the high resolution separation of reducing sugars labeled with the fluorophore 8-aminonaphthalene-1,3, 6-trisulfonic acid. Biochem. J. 270, 705–713.PubMedGoogle Scholar
  3. 3.
    Jackson, P. and Williams, G. R. (1991) Polyacrylamide gel electrophoresis of reducing saccharides labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid: application to the enzymatic and structural analysis of oligosaccharides. Electrophoresis 12, 94–96.PubMedCrossRefGoogle Scholar
  4. 4.
    Jackson, P. (1994) The analysis of fluorophore-labeled glycans by high-resolution polyacrylamide gel electrophoresis. Analyt. Biochem. 216, 243–252.PubMedCrossRefGoogle Scholar
  5. 5.
    Starr, C., Masada, R. I., Hague, C., Skop, E., and Klock, J. (1996) Fluorophore-assisted-carbohydrate-electrophoresis, FACE® in the separation, analysis, and sequencing of carbohydrates. J. Chromatogr. A 720, 295–321.PubMedCrossRefGoogle Scholar
  6. 6.
    Masada, R. I., Hague, C., Seid, R., Ho, S., McAlister, S., Pigiet, V., and Starr, C. (1995) Fluorophore-assisted-carbohydrate-electrophoresis, FACE®, for determining the nature and consistency of recombinant protein glycosylation,. Trends Glycosci. Glycotech. 7, 133–147.Google Scholar
  7. 7.
    Hu, G. (1995) Fluorophore assisted carbohydrate electrophoresis technology and applications. J. Chromatogr. 705, 89–103CrossRefGoogle Scholar
  8. 8.
    Higgins, E. and Friedman, Y. (1995) A method for monitoring the glycosylation of recombinant glycoproteins from conditioned medium, using fluorophore assisted carbohydrate electrophoresis. Analyt. Biochem. 228, 221–225.PubMedCrossRefGoogle Scholar
  9. 9.
    Basu, S. S., Dastgheib-Hosseini, S., Hoover, G., Li, Z., and Basu, S. (1994) Analysis of glycosphingolipids by fluorophore-assisted carbohydrate electrophoresis using ceramide glycanase from Mercenaria mercenaria. Analyt. Biochem. 222, 270–274.PubMedCrossRefGoogle Scholar
  10. 10.
    Flesher, A. R., Marzowski, J, Wang, W., and Raff, H. V. (1995) Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion proteins: correlation of oligosaccharide content with in vivo clearance profile. Biotech. Bioeng. 46, 399–407.CrossRefGoogle Scholar
  11. 11.
    Lee, K. B., Al-Hakim, A., Loganathan, D., and Linhard, R. J. (1991) A new method for sequencing carbohydrates using charged and fluorescent conjugates. Carbohydr. Res. 214, 155–162.PubMedCrossRefGoogle Scholar
  12. 12.
    Stack, R. J. and Sullivan, M. T. (1992) Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1, 3,6-trisulfonic acid. Glycobiology 2, 85–92.PubMedCrossRefGoogle Scholar
  13. 13.
    Roy, S. N., Kudryk, B., and Redman, C. M. (1995) Secretion of biologically active recombinant fibrinogen by yeast. J. Biol. Chem. 270, 23,761–23,767.PubMedCrossRefGoogle Scholar
  14. 14.
    Denny, P. C., Denny, P. A., and Hong-Le, N. H. (1995) Asparagine-linked oligosaccharides in mouse mucin. Glycobiology 5, 589–597.PubMedCrossRefGoogle Scholar
  15. 15.
    Qu, Z., Sharkey, R. M., Hansen, H. J., Goldenberg, D. M., and Leung, S.-O. (1997) Structure determination of N-linked oligosaccharides engineered at the CH1 domain of humanized LL2. Glycobiology 7, 803–809.PubMedCrossRefGoogle Scholar
  16. 16.
    Prieto, P. A., Mukerji, P., Kelder, B., Erney, R., Gonzalez, D., Yun, J. S., et al. (1995) Remodeling of mouse milk glycoconjugates by transgenic expression of a human glycosyltransferase. J. Biol. Chem. 270, 29,515–29,519.PubMedCrossRefGoogle Scholar
  17. 17.
    Pirie-Shepherd, S., Jett, E. A., Andon, N. L., and Pizzo, S. V. (1995) Sialic acid content of plasminogen 2 glycoforms as a regulator of fibrinolytic activity. J. Biol. Chem. 270, 5877–5881.PubMedCrossRefGoogle Scholar
  18. 18.
    Goss, P. E., Baptiste, J., Fernandes, B., Baker, M., and Dennis, J. W. (1994) A phase I study of swainsonine in patients with advanced malignancies. Cancer Res. 54, 1450–1457.PubMedGoogle Scholar
  19. 19.
    Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C. J., Skop, E., et al. (1996) Mice lacking both subunits of lysosomal beta-hexosaminidase display gangliosidosis and mucopolysaccharidosis. Nature Genet. 14, 348–352.PubMedCrossRefGoogle Scholar
  20. 20.
    Masada, R. I., Skop, E., and Starr, C. M. (1996) Fluorophore-assisted-carbohydrate-elec-trophoresis for quality control of recombinant protein glycosylation. Biotechnol. Appl. Biochem. 24, 195–205.Google Scholar
  21. 21.
    Kumar, H. P. M., Hague, C., Haley, T., Starr, C. M., Besman, M. J., Lundblad, R., and Baker, D. (1996) Elucidation of N-linked oligosaccharide structures of recombinant factor VII using fluorophore assisted carbohydrate electrophoresis. Biotechnol. Appl. Biochem. 24, 207–216.PubMedGoogle Scholar
  22. 22.
    Tsuchida, F., Tanaka, T., Matuo, Y., Moriyama, N., Okada, K., Jimbo, H., et al. (1997) Oligosaccharide Analysis Of Renal Cell Carcinoma by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). Jpn J. Electroph. 41, 39–41, 81-83.Google Scholar
  23. 23.
    Hague, C., Masada, R. I., and Starr, C. (1998) Structural determination of oligosaccharides from recombinant iduronidase released with peptide N-glycase F, using Fluorophore-Assisted Carbohydrate Electrophoresis. Electrophoresis 19, 2612–2620PubMedCrossRefGoogle Scholar
  24. 24.
    Morimoto, K., Maeda, N., Foad, A., Toyoshima, S., and Hayakawa, T. (1999) Structural Characterization of Recombinant Human Erythropoietins by Fluorophore-assisted Carbohydrate Electrophoresis. Biol. Pharm. Bull. 22, 5–10.PubMedGoogle Scholar
  25. 25.
    Allen, A. C., Bailey, E. M., Barratt, J., Buck, K. S., and Feehally, J. (1999) Analysis of IgA1 O-glycans in IgA nephropathy by fluorophore-assisted carbohydrate electrophoresis. J. Am. Soc. Nephrol. 10, 1763–1771.PubMedGoogle Scholar
  26. 26.
    Bardor, M., Cabanes-Macheteau, M., Faye, L., and Lerouge, P. (2000) Monitoring the N-glycosylation of plant glycoproteins by fluorophore-assisted carbohydrate electrophoresis. Electrophoresis. 21, 2250–2256.CrossRefGoogle Scholar
  27. 27.
    Calabro, A., Benavides, M., Tammi, M., Hascall, V. C., and Midura, R. J., (2000) Microanalysis of enzyme digests hyaluronan and chondroitin/dermatan sulfate by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE). Glycobiology 10, 273–281.PubMedCrossRefGoogle Scholar
  28. 28.
    Calabro, A. C., Hascall, V. J., and Midura, R., (2000) Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using Fluorophore-Assisted Carbohydrate Electrophoresis. Electrophoresis 21, 2296–2308.CrossRefGoogle Scholar
  29. 29.
    Frado, L. Y. and Strickler, J. E. (2000) Relative abundance of oligosaccharides in candida species as determined by Fluorophore-Assisted Carbohydrate Electrophoresis. J. Clin. Microbiol. 38, 2862–2869.Google Scholar
  30. 31.
    Lemoine, J., Cabanes-Macheteau, M., Bardor, M., Michalaski, J. C., Faye, L., and Lerouge, P. (2000) Analysis of 8-aminoapthalene-1,3,6-trisulfonic acid labelled N-glycans by matrix assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun Mass Spectrom, 14, 100–104.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Brian K. Brandley
    • 1
  • John C. Klock
  • Christopher M. Starr
    • 1
  1. 1.Glyko Inc.Navato

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