Characterization of N-linked oligosaccharides by electrospray and tandem mass spectrometry.
Electrospray and tandem mass spectrometry are used to characterize underivatized oligosaccharides that have been digested from asparagine side chains of glycoproteins. Oligosaccharides that contain sialic acids were detected with the best sensitivity in the negative-ion detection mode whereas those that do not contain sialic acid were detected with the best sensitivity in the positive-ion detection mode. The positive-ion abundances of oligosaccharides were greatly enhanced in electrospray mass spectra by adding 10 mM sodium acetate or ammonium acetate to the sample solvent. Tandem mass spectrometry was used to determine primary structural features of the oligosaccharides. Methodology that has been developed on branched high-mannose, hybrid, and complex carbohydrate standards was applied to a mixture of oligosaccharides that were digested with N-glycanase from the glycoprotein, ovalbumin. The composition and relative abundances of individual oligosaccharides obtained from the electrospray mass spectrum compare favorably to those obtained by anion-exchange chromatography/pulsed amperometric detection and by gel permeation chromatography of the oligosaccharides after radiolabelling the reducing end of the carbohydrates. The oligosaccharide content of ovalbumin was independently determined from the heterogeneity observed in the electrospray mass spectrum of the intact 44-kDa glycoprotein. Comparison of the oligosaccharide compositions determined before and after enzymatic digestion shows a selective digestion of high-mannose and low molecular weight oligosaccharides by N-glycanase.
Duffin
K L
KL
Monsanto Corporate Research, Monsanto Company, St. Louis, Missouri 63198.
Welply
J K
JK
Huang
E
E
Henion
J D
JD
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D013058:Mass Spectrometry / Q000379:methods* / Q000592:standards; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*
D009844:Oligosaccharides