Identification and Characterization of Protein Glycosylation Using Specific Endo- and Exoglycosidases

  • Paula MagnelliEmail author
  • Alicia Bielik
  • Ellen Guthrie
Part of the Methods in Molecular Biology book series (MIMB, volume 801)


Enzymatic deglycosylation followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples. Specific glycosidases were used to remove sugars from glycoproteins in a controlled fashion leaving the protein core intact; the resulting change in molecular weight could be detected as shifts in gel mobility. Alternatively, glycan-sensitive reagents were used to visualize the intensity of glycoprotein bands before and after enzyme treatment. The ease of use of these techniques, which require only basic laboratory instrumentation and reagents, makes them the methodology of choice for initial glycobiology studies. These protocols are also well suited to screen for optimal expression conditions, since multiple glycoprotein samples can be processed at once.

Key words

Glycoprotein N-Glycan O-Glycan O-GlcNAc PNGase F O-Glycosidase Deglycosylation 


  1. 1.
    Rich JR, Withers SG. Emerging methods for the production of homogeneous human glycoproteins. Nat Chem Biol. 2009; 5(4):206–15.PubMedCrossRefGoogle Scholar
  2. 2.
    Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology. 2009; 19(9):936–49.PubMedCrossRefGoogle Scholar
  3. 3.
    Wells L, Vosseller K, Hart GW. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science. 2001, 291(5512):2376–8.PubMedCrossRefGoogle Scholar
  4. 4.
    Butkinaree C, Park K, Hart GW. O-linked β-N-acetylglucosamine (O-GlcNAc): Extensive crosstalk with phosphorylation to regulate signaling and transcription in response to nutrients and stress. Biochim Biophys Acta. 2010; 1800(2):96–106.PubMedCrossRefGoogle Scholar
  5. 5.
    Love DC, Hanover JA. The hexosamine signaling pathway: deciphering the “O-GlcNAc code”. Sci STKE. 2005; 2005(312): re13.Google Scholar
  6. 6.
    Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci Signal. 2010, 3(104): ra2.Google Scholar
  7. 7.
    Lazarus BD, Love DC, Hanover JA. O-GlcNAc cycling: implications for neurodegenerative disorders. Int J Biochem Cell Biol. 2009, 41(11):2134–46PubMedCrossRefGoogle Scholar
  8. 8.
    Bielik AM, Zaia J. Extraction of chondroitin/dermatan sulfate glycosaminoglycans from connective tissue for mass spectrometric analysis. Methods Mol Biol. 2010, 600:215–25.PubMedCrossRefGoogle Scholar
  9. 9.
    Azzouz N, Gerold P, Schwarz RT. Metabolic labeling and structural analysis of glycosylphosphatidylinositols from parasitic protozoa. Methods Mol Biol. 2008, 446:183–98.PubMedCrossRefGoogle Scholar
  10. 10.
    Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME. (Eds). Essentials of Glycobiology, 2nd ed. 2008. Cold Spring Harbor Laboratory Press. Plainview (NY).Google Scholar
  11. 11.
    Carlsen RB, Bahl OP, Swaminathan N. Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit. J Biol Chem. 1973, 248(19):6810–2.Google Scholar
  12. 12.
    Lectin analysis of proteins blotted onto filters. Freeze HH. Curr Protoc Mol Biol. 2001; Chapter 17:Unit17.7.Google Scholar
  13. 13.
    Thakur D, Rejtar T, Karger BL, Washburn NJ, Bosques CJ, Gunay NS, Shriver Z, Venkataraman G. Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry. Anal Chem. 2009, 81(21):8900–7.PubMedCrossRefGoogle Scholar
  14. 14.
    Comer FI, Vosseller K, Wells L, Accavitti MA, Hart GW. Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal Biochem. 2001; 293(2):169–77.PubMedCrossRefGoogle Scholar
  15. 15.
    Boeggeman E, Ramakrishnan B, Kilgore C, Khidekel N, Hsieh-Wilson LC, Simpson JT, Qasba PK. Direct identification of nonreducing GlcNAc residues on N-glycans of glycoproteins using a novel chemoenzymatic method. Bioconjug Chem. 2007; 18(3):806–14.PubMedCrossRefGoogle Scholar
  16. 16.
    Roquemore EP, Dell A, Morris HR, Panico M, Reason AJ, Savoy LA, Wistow GJ, Zigler JS Jr, Earles BJ, Hart GW. Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine. Journal of Biological Chem. 1992, 267:555–63.Google Scholar
  17. 17.
    Bielik, A.M. New England Biolabs, Inc., Unpublished Results.Google Scholar
  18. 18.
    Clark PM, Dweck JF, Mason DE, Hart CR, Buck SB, Peters EC, Agnew BJ, Hsieh-Wilson LC. Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J Am Chem Soc. 2008; 130(35):11576–7.PubMedCrossRefGoogle Scholar
  19. 19.
    Kelly WG, Hart GW. Glycosylation of chromosomal proteins: localization of O-linked N-acetylglucosamine in Drosophila chromatin. Cell. 1989; 57(2):243–51.PubMedCrossRefGoogle Scholar
  20. 20.
    Zachara NE. Detection and analysis of O-linked β-N-acetylglucosamine-modified proteins. Methods Mol Biol. 2009; 464:227–54.PubMedCrossRefGoogle Scholar
  21. 21.
    Park S, Lee MR, Shin I Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes. Chem Commun (Cambridge). 2008;(37):4389–99.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.New England BiolabsIpswichUSA

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