1H NMR characterization of a hen ovalbumin tyrosinamide N-linked oligosaccharide library.
A library of 15 N-linked oligosaccharide structures was prepared from ovalbumin and characterized using high-field NMR and mass spectrometry. The oligosaccharides were enzymatically released from ovalbumin glycopeptides, and the reducing ends were reacted with ammonium bicarbonate to form oligosaccharide-glycosylamines. These reacted with Boc-tyrosine-N-hydroxysuccinimide ester, resulting in a mixture of tyrosinamide-oligosaccharides. The Boc group was removed to expose an amine terminus which enhanced the resolution of tyrosinamide-oligosaccharides when chromatographed on reverse-phase HPLC. Ten major and five minor oligosaccharides were purified on a micromole scale and characterized using 1H NMR and FAB-MS. The structures include high-mannose, hybrid, and complex oligosaccharides possessing from two to five antenna, providing the most complete definition of ovalbumin N-linked oligosaccharides to date. The resulting library is well suited to biological studies due to the presence of a single terminal tyrosine residue on each oligosaccharide that allows radioiodination or the attachment of additional probes to these glycoconjugates prior to biological studies.
Corradi Da Silva
M L
ML
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus 43210, USA.
Stubbs
H J
HJ
Tamura
T
T
Rice
K G
KG
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D005260:Female; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010047:Ovalbumin / Q000737:chemistry*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin; D014443:Tyrosine / Q000031:analogs & derivatives*
D006020:Glycopeptides; D009844:Oligosaccharides; C036387:tyrosinamide; D014443:Tyrosine; D006859:Hydrogen; D010047:Ovalbumin; D014357:Trypsin