Selective organic precipitation/extraction of released N-glycans following large-scale enzymatic deglycosylation of glycoproteins.
A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N(4)-(N-acetyl-beta-glucosaminyl) Asn amidase (EC 3.5.1. 52)-released complex, and beta-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at -20 degrees C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.
Verostek
M F
MF
Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509, USA.
Lubowski
C
C
Trimble
R B
RB
D000818:Animals; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification*
D006023:Glycoproteins; D011134:Polysaccharides