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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

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Abstract

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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Author information

Affiliations

  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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https://doi.org/10.1038/nprot.2008.76

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