title: A method for monitoring the glycosylation of recombinant glycoproteins from conditioned medium, using fluorophore-assisted carbohydrate electrophoresis.
abstract: We have developed a method for monitoring the N-glycosylation of recombinant glycoproteins directly from conditioned medium samples. Proteins in the conditioned medium are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After staining the membranes with Coomassie blue, the protein(s) of interest is excised. Oligosaccharides are released from the membrane-bound glycoprotein by digesting with peptide N4-(acetyl-beta-glucosaminyl) asparagine amidase and labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS). Labeled oligosaccharides are then separated on polyacrylamide gels which allow for the direct comparison of samples. We have shown that recombinant human lysosomal hydrolase alpha-galactosidase A is N-glycosylated with both sialylated and phosphorylated oligosaccharides. ANTS-labeled oligosaccharide bands from alpha-galactosidase A were isolated from polyacrylamide gels. Sialylated and phosphorylated bands were identified by shifts in their electrophoretic mobility after digesting with neuraminidase or alkaline phosphatase to remove sialic acid or phosphate groups, respectively. Using the ANTS-labeled oligosaccharides from alpha-galactosidase A, we have shown that polyacrylamide gels can be used to resolve sialylated and phosphorylated oligosaccharide structures.
authors.affiliation: Genzyme Corporation, Framingham, Massachusetts 01701-9322, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Friedman
authors.forename: E A
authors.initials: EA
authors.lastname: Higgins
mesh_terms: D000581:Amidohydrolases; D002241:Carbohydrates / Q000032:analysis*; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006596:Hexosaminidases; D006801:Humans; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009844:Oligosaccharides / Q000032:analysis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011145:Polyvinyls; D011994:Recombinant Proteins / Q000032:analysis; D012394:Rosaniline Dyes; D000519:alpha-Galactosidase / Q000737:chemistry; D005465:Fluorocarbon Polymers
chemical_list: D000581:Amidohydrolases; D002241:Carbohydrates; D017077:Culture Media, Conditioned; D006023:Glycoproteins; D006596:Hexosaminidases; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009844:Oligosaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011145:Polyvinyls; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; D000519:alpha-Galactosidase; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C048139:Coomassie blue