Incorporation of Unnatural Sugars for the Identification of Glycoproteins

  • Balyn W. Zaro
  • Howard C. Hang
  • Matthew R. PrattEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 951)


Glycosylation is an abundant post-translational modification that alters the fate and function of its substrate proteins. To aid in understanding the significance of protein glycosylation, identification of target proteins is key. As with all proteomics experiments, mass spectrometry has been established as the desired method for substrate identification. However, these approaches require selective enrichment and purification of modified proteins. Chemical reporters in combination with bioorthogonal reactions have emerged as robust tools for identifying post-translational modifications including glycosylation. We provide here a method for the use of bioorthogonal chemical reporters for isolation and identification of glycosylated proteins. More specifically, this protocol is a representative procedure from our own work using an alkyne-bearing O-GlcNAc chemical reporter (GlcNAlk) and a chemically cleavable azido-azo-biotin probe for the identification of O-GlcNAc-modified proteins.

Key words

Proteomics Glycosylation Bioorthogonal chemical reporter Click chemistry Azide Mass spectrometry O-GlcNAc 



We thank Leslie Bateman for careful reading of the manuscript. This work was supported by the University of Southern California and the American Cancer Society Grant IRG-58-007-51 (to M.R.P.) and the Ellison Medical Foundation and the National Institutes of Health and General Medical Sciences Grant 1RO1GM087544 O1A2 (to H.C.H.).


  1. 1.
    Zielinska DF, Gnad F, Wisniewski JR, Mann M (2010) Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 141(5):897–907CrossRefPubMedGoogle Scholar
  2. 2.
    Teo CF et al (2010) Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6(5):338–343CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Saxon E, Bertozzi CR (2000) Cell surface engineering by a modified Staudinger reaction. Science 287(5460):2007–2010CrossRefPubMedGoogle Scholar
  4. 4.
    Hang HC, Yu C, Kato DL, Bertozzi CR (2003) A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation. Proc Natl Acad Sci USA 100(25):14846–14851CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Dehnert KW et al (2011) Metabolic labeling of fucosylated glycans in developing zebrafish. ACS Chem Biol 6(6):547–552CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Dube DH, Prescher JA, Quang CN, Bertozzi CR (2006) Probing mucin-type O-linked glycosylation in living animals. Proc Natl Acad Sci USA 103(13):4819–4824CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Zaro BW, Bateman LA, Pratt MR (2011) Robust in-gel fluorescence detection of mucin-type O-linked glycosylation. Bioorg Med Chem Lett 21(17):5062–5066Google Scholar
  8. 8.
    Hanson SR et al (2007) Tailored glycoproteomics and glycan site mapping using saccharide-selective bioorthogonal probes. J Am Chem Soc 129(23):7266–7267CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Boyce M et al (2011) Metabolic cross-talk allows labeling of O-linked {beta}-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway. Proc Natl Acad Sci USA 108(8):3141–3146CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zaro BW, Yang Y-Y, Hang HC, Pratt MR (2011) Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proc Natl Acad Sci USA 108(20):8146–8151Google Scholar
  11. 11.
    Sprung R et al (2005) Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J Proteome Res 4(3):950–957CrossRefPubMedGoogle Scholar
  12. 12.
    Nandi A et al (2006) Global identification of O-GlcNAc-modified proteins. Anal Chem 78(2):452–458CrossRefPubMedGoogle Scholar
  13. 13.
    Clark PM et al (2008) Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. J Am Chem Soc 130(35):11576–11577CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wang Z et al (2010) Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Mol Cell Proteomics 9(1):153–160CrossRefPubMedGoogle Scholar
  15. 15.
    Wang Z et al (2010) Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Science STKE 3(104):ra2Google Scholar
  16. 16.
    Yang Y-Y, Ascano JM, Hang HC (2010) Bioorthogonal chemical reporters for monitoring protein acetylation. J Am Chem Soc 132(11):3640–3641CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Fonović M, Verhelst SHL, Sorum MT, Bogyo M (2007) Proteomics evaluation of chemically cleavable activity-based probes. Mol Cell Proteomics 6(10):1761–1770CrossRefPubMedGoogle Scholar
  18. 18.
    Verhelst SHL, Fonović M, Bogyo M (2007) A mild chemically cleavable linker system for functional proteomic applications. Angew Chem Int Ed 46(8):1284–1286CrossRefGoogle Scholar
  19. 19.
    Denny JB, Blobel G (1984) 125I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage. Proc Natl Acad Sci USA 81(17):5286–5290CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Gurcel C et al (2008) Identification of new O-GlcNAc modified proteins using a click-chemistry-based tagging. Anal Bioanal Chem 390(8):2089–2097CrossRefPubMedGoogle Scholar
  21. 21.
    Saxon E et al (2002) Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J Am Chem Soc 124(50):14893–14902CrossRefPubMedGoogle Scholar
  22. 22.
    Rabuka D, Hubbard SC, Laughlin ST, Argade SP, Bertozzi CR (2006) A chemical reporter strategy to probe glycoprotein fucosylation. J Am Chem Soc 128(37):12078–12079CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Hsu T-L et al (2007) Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci USA 104(8):2614–2619CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Goon S, Schilling B, Tullius MV, Gibson BW, Bertozzi CR (2003) Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides. Proc Natl Acad Sci USA 100(6):3089–3094CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Balyn W. Zaro
    • 1
  • Howard C. Hang
    • 2
  • Matthew R. Pratt
    • 1
    • 3
    Email author
  1. 1.Department of ChemistryUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.The Laboratory of Chemical Biology and Microbial PathogenesisThe Rockefeller UniversityNew YorkUSA
  3. 3.Department of Molecular and Computational BiologyUniversity of Southern CaliforniaLos AngelesUSA

Personalised recommendations