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Use of high-performance anion exchange chromatography with pulsed amperometric detection for O-glycan determination in yeast

Nature Protocols volume 3, pages 10261031 (2008) | Download Citation



O-glycosylation is a post-translational protein modification that occurs in all eukaryotes. Yeasts have received increasing attention as a host for therapeutic protein production because of their ability to secrete high levels of recombinant protein. Because yeasts such as Pichia pastoris have been shown to O-glycosylate some proteins with varying effects on protein function, it is important to elucidate the nature of this modification. Methods that characterize O-glycosylation on a qualitative and quantitative basis are thus important when considering yeast as a host for therapeutic protein production. This protocol describes the release of O-glycans from a protein sample by β-elimination under alkaline conditions using sodium borohydride and sodium hydroxide. The released O-linked oligosaccharides are subsequently processed and then separated by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). An estimation of O-glycan molar occupancy and average O-mannose chain length is ultimately derived. This protocol requires 3 d for completion. This method provides an assessment of O-glycosylation and allows one to correlate the effect of O-glycosylation on protein properties.

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  1. 1.

    , , & Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23–52 (2000).

  2. 2.

    Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat. Biotechnol. 22, 1409–1414 (2004).

  3. 3.

    Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43R–56R (2002).

  4. 4.

    et al. Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA 100, 5022–5027 (2003).

  5. 5.

    et al. Production of complex human glycoproteins in yeast. Science 301, 1244–1246 (2003).

  6. 6.

    et al. Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313, 1441–1443 (2006).

  7. 7.

    & The humanization of N-glycosylation pathways in yeast. Nat. Rev. Microbiol. 3, 119–128 (2005).

  8. 8.

    , , , & O-mannosyl glycans: from yeast to novel associations with human disease. Curr. Opin. Struct. Biol. 13, 621–630 (2003).

  9. 9.

    et al. Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris. Glycobiology 14, 265–274 (2004).

  10. 10.

    et al. Expression and characterization of recombinant human antithrombin III in Pichia pastoris. Protein Expr. Purif. 23, 55–65 (2001).

  11. 11.

    , & Site-specific O-glycosylation of human granulocyte/macrophage colony-stimulating factor secreted by yeast and animal cells. Eur. J. Biochem. 203, 663–667 (1992).

  12. 12.

    , & Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass Spectrom. 15, 659–672 (2004).

  13. 13.

    , & Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis. Anal. Chem. 73, 6063–6069 (2001).

  14. 14.

    , , , & Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis. Glycobiology 15, 747–775 (2005).

  15. 15.

    et al. Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal. Biochem. 230, 229–238 (1995).

  16. 16.

    et al. The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc alpha receptor interactions. J. Biol. Chem. 273, 2260–2272 (1998).

  17. 17.

    et al. Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry 32, 679–693 (1993).

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  1. Analytical Development GlycoFi, 21 Lafayette Street, Suite 200, Lebanon, New Hampshire 03766, USA.

    • Terrance A Stadheim
    • , Huijuan Li
    •  & Irina N Burnina
  2. Glycan Biosciences, Level 1, 66 St. Georges Terrace, Perth, Western Australia, Australia 6000.

    • Warren Kett
  3. Department of Biology, Dartmouth College, Thayer School of Engineering, Hanover, New Hampshire 03766, USA.

    • Tillman U Gerngross
  4. Department of Chemistry, Dartmouth College, Thayer School of Engineering, Hanover, New Hampshire 03766, USA.

    • Tillman U Gerngross


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Correspondence to Tillman U Gerngross.

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