Carbohydrate analysis of glycoproteins generally begins with the release of intact oligosaccharides from the protein polypeptide chain by specific endoglycosidases or chemical treatments. The oligosaccharides are then subjected to monosaccharide/oligosaccharide compositional analysis by either: chromatographic analysis (such as Dionex chromatography and gas chromatography), mass spectrometry, electrophoresis or a combination of these methods.
However there are problems associated with the analysis of oligosaccharides released from glycoproteins that involve the loss of positional information, such as:
(a) where are the oligosaccharides are conjugated on the polypeptide chain?
(b) is there site specific heterogeneity?
(c) how much sugar is there at each site of glycosylation?
Two techniques have revolutionised the traditional approach to oligosaccharide analysis of glycoproteins: electrospray mass spectrometry and solid-phase Edman degradation. The coupling of HPLC to a mass spectrometer via the electrospray interface has allowed the rapid isolation and characterisation of glycopeptides using the source induced fragmentation method of Carr [1]. The second approach is by solidphase Edman degradation, a chemical method, for the site specific quantitative and qualitative identification and characterisation of glycoamino acids recovered during the course of normal Edman sequencing.
We have combined the techniques of HPLC/ESMS and solid-phase degradation to rapidly identify and recover glycopeptides and glycoamino acids. We have illustrated the complementary nature of this powerful approach with the site specific characterisation of the glycoamino acids on the glycoprotein Bovine Fetuin.