Use of high-performance anion exchange chromatography with pulsed amperometric detection for O-glycan determination in yeast.
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 approximately 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.
Terrance A
TA
Stadheim
Huijuan
H
Li
Warren
W
Kett
Irina N
IN
Burnina
Tillman U
TU
Gerngross
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D009005:Monosaccharides / Q000032:analysis; D009172:Mycology / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015003:Yeasts / Q000737:chemistry* / Q000378:metabolism
D005656:Fungal Proteins; D006023:Glycoproteins; D009005:Monosaccharides; D011134:Polysaccharides