The covalent structure of individual N-linked glycopeptides from ovomucoid and asialofetuin.
In order to explore whether individual N-linked glycans in a given glycoprotein may be processed to different end products and at the same time prepare a number of well characterized glycopeptides as substrates for glycopeptide hydrolases, we have prepared the individual glycopeptides representing the four major glycosylation sites in ovomucoid and the three sites in asialofetuin. The individual glycopeptides were characterized by amino acid sequence determination before and after removal of the glycan by peptide:N-glycanase (amidase), and the liberated glycans were subjected to mass spectrometric analysis. As expected from available sugar analyses of the individual glycans in ovomucoid, no major differences were detected between the four glycosylation sites in this glycoprotein, but a definite trend toward less processed (less extensively branched) species was observed in going from site 1 to 4. In fetuin, for which the glycan pool is known to be made up of about two-thirds triantennary and one-third biantennary structures, the analysis of the three glycopeptides gave triantennary to biantennary ratios of 75/25, 67/33, and 70/30, respectively, demonstrating that the three sites are processed to a very similar, albeit perhaps not identical, extent. All the glycopeptides obtained in these studies, including the CNBr-produced glycopeptide from ovalbumin, were purified by a set series of steps, gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DE52 and/or reverse phase high performance liquid chromatography. Based on the results, these procedures appear to have general application for the preparation of glycopeptides.
D000596:Amino Acids / Q000032:analysis; D001212:Asialoglycoproteins; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D004527:Egg Proteins; D060748:Fetuins; D006020:Glycopeptides / Q000302:isolation & purification; D013058:Mass Spectrometry; D010059:Ovomucin; D010446:Peptide Fragments / Q000302:isolation & purification; D011402:Pronase; D014357:Trypsin; D000509:alpha-Fetoproteins; D001616:beta-Galactosidase
D000596:Amino Acids; D001212:Asialoglycoproteins; D004527:Egg Proteins; D060748:Fetuins; D006020:Glycopeptides; D010446:Peptide Fragments; D000509:alpha-Fetoproteins; C017029:asialofetuin; D010059:Ovomucin; D001616:beta-Galactosidase; D002918:Chymotrypsin; D014357:Trypsin; D011402:Pronase; D003488:Cyanogen Bromide