Analysis of N-linked oligosaccharides: progress towards the characterisation of glycoprotein-linked carbohydrates.
The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease.
Charlwood
J
J
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North), Harlow, Essex, UK.
Bryant
D
D
Skehel
J M
JM
Camilleri
P
P
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis* / Q000302:isolation & purification
D006023:Glycoproteins; D007074:Immunoglobulin G; D009844:Oligosaccharides