Protocol abstract
Nature Protocols 3, - 1026 - 1031 (2008)
Published online: 22 May 2008 | doi:10.1038/nprot.2008.76
Subject Categories: Biochemistry and protein analysis | Isolation, purification and separation
Use of high-performance anion exchange chromatography with pulsed amperometric detection for O-glycan determination in yeast
Terrance A Stadheim1, Huijuan Li1, Warren Kett2, Irina N Burnina1 & Tillman U Gerngross3,4
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 
-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.
- Analytical Development GlycoFi, 21 Lafayette Street, Suite 200, Lebanon, New Hampshire 03766, USA.
- Glycan Biosciences, Level 1, 66 St. Georges Terrace, Perth, Western Australia, Australia 6000.
- Department of Biology, Dartmouth College, Thayer School of Engineering, Hanover, New Hampshire 03766, USA.
- Department of Chemistry, Dartmouth College, Thayer School of Engineering, Hanover, New Hampshire 03766, USA.
Correspondence to: Tillman U Gerngross3,4 e-mail: tillman.gerngross@dartmouth.edu
nature-products
A-Z product listing
- 0.4 M NaOH/2.5 M NaBH4 (Sigma)
- 1 M NaOH (prepared from 50% wt/wt NaOH(Burdick & Jackson)
- 1.0-ml Glass conical vials in loader(Biotech Solutions)
- AG 50W-X8, H+ form [50% (vol/vol) slurry in 0.1% acetic acid(Bio-Rad)
- Amicon ultra centrifugal filter devices(Millipore)
- Carbopac MA1 250
4 mm2 column fitted with 50 4 mm2 amino trap column(Dionex)
MORE ARTICLES LIKE THIS
These links to content published by NPG are automatically generated.
NEWS AND VIEWS
Sweet control of cell migration, cytokinesis and organogenesisNature Cell Biology News and Views (01 Jan 2004)
