Carbohydrate analysis of glycoproteins: from pvdf membranes to maldi/cid

Glycoproteins on PVDF membranes have been analyzed for monosaccharide composition and by oligosaccharide mapping using high-pH anion-exchange chromatography with pulsed amperometric detection (Weitzhandler et al., (1993) J. Biol. Chem. 268, 5121). More recently, labeling of released monosaccharides and oligosaccharides with a chromatographicand mass spectrometric-compatible fluorophore has increased sensitivity 100-fold. A novel strategy has been developed for the structural analysis of protein glycosylation (N-linked) from electroblotted Coomassie-stained bands which involves: i) releasing oligosaccharide classes from the immobilized glycoprotein with specific endoglycosidases and then with PNGase F; ii) labeling the reducing end with 2-aminobenzamide; iii) separating oligosaccharides using multi-mode hydrophilic interaction chromatography with fluorometric detection and iv) analyzing using matrix-assisted laser desorption mass spectrometry with collision-induced dissociation. Carbohydrate analysis of an electro-blotted erythrocyte water channel (CHIP28), parietal cell anion exchanger (AE2) and the H,K ATPase, both from the same cell type, will be presented.