ISSN:
0173-0835
Keywords:
Erythropoietin
;
Fetuin
;
Kallikrein
;
Ribonuclease B
;
8-Amino-1,3,6-trisulfonic acid
;
Sialidase
;
Capillary electrophoresis
;
Laser-induced fluoresence detection
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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 resoluton of the N-linked oligosaccharide profile for fingerprinting glycans derived from glycoproteins.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150191512
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