Selective organic precipitation/extraction of released N-glycans followinglarge-scale enzymatic deglycosylation of glycoproteins

A major difficulty with isolating enzymatically or chemically released olig osaccharides 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 develop ed a rapid organic solvent precipitation/extraction procedure that allows s equential isolation of endo-beta=N-acetylglucosaminidase H (EC 3.2.1.96)-re leased high-mannose and hybrid, peptide-N-4-(N-acetyl-beta-glucosaminyl) As n amidase (EC 3.5.1.52)-released complex, and beta-eliminated O-linked glyc ans without the need for intermediate chromatography, desalting, or concent ration steps. The method involves precipitation of protein and released gly cans at -20 degrees C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three po ols of essentially salt-and detergent-free oligosaccharides (high-mannose/h ybrid, complex, and O-linked) can be isolated in a high yield in 4 days wit h this protocol, which has been extensively tested using bovine RNase B, hu man bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin , bovine fetuin, bovine thyroglobulin, and several invertase preparations f rom wild-type and mutant yeast strains. (C) 2000 Academic Press.