Profiling glycoprotein n-linked oligosaccharide by capillary electrophoresis.
A method for analysis of N-linked oligosaccharides derived from glycoproteins including sialic acid-containing species is presented. It is based on the combination of specific chemical and enzymatic conversions coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. Glycoproteins were heat-denatured in the presence of a reducing agent and the N-linked oligosaccharides were released by peptide N-glycosidase (PNGase F; EC3.5.1.52)-catalyzed hydrolysis. The released N-linked oligosaccharides were derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) under mild reductive amination conditions in which desialylation and loss of fucose residues are minimized. A model N-linked oligosaccharide, desialylated, galactosylated biantennary, core-substituted with fucose (A2F) was tested for APTS-based derivatization chemistry with excellent recovery of the adduct without losing fucose and neuraminic acid residues. The profiles of heavily sialylated N-linked oligosaccharides derived from fetuin, recombinant human erythropoietin and kallikrein are reported and the data show that the present method produces a high resolution of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.
Chen
F T
FT
Beckman Coulter Inc., Fullerton, CA 92835, USA. afchen@beckman.com
Evangelista
R A
RA
D000581:Amidohydrolases; D002240:Carbohydrate Sequence; D002241:Carbohydrates; D002384:Catalysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis; D005456:Fluorescent Dyes; D005936:Glucans; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007610:Kallikreins / Q000032:analysis; D008320:Maltose; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis*; D010084:Oxidation-Reduction; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011721:Pyrenes; D012260:Ribonucleases / Q000032:analysis; D000509:alpha-Fetoproteins / Q000032:analysis
C103430:8-aminopyrene-1,3,6-trisulfonic acid; D002241:Carbohydrates; D005456:Fluorescent Dyes; D005936:Glucans; D006023:Glycoproteins; D009844:Oligosaccharides; D011721:Pyrenes; D000509:alpha-Fetoproteins; D004921:Erythropoietin; C031146:maltoheptaose; D008320:Maltose; D012260:Ribonucleases; C029600:ribonuclease B; D007610:Kallikreins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase