Rapid characterization of asparagine‐linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer

Abstract

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide Nglycosidase F, endo-P-N-acetylglucosaminidase F and endo-P-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anionexchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1 -0.3 pA output with suitable sensitivity (< 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients.Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestions (> 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ionexchange chromatography of the aqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and al-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri-and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with a-mannosidase. Treatment with P-galactosidase, P-N-acetylglucosaminidase and a-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with a-L-fucosidase, endo-P-N-acetylglucosaminidase F and endo-P-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue.Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endoand exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.Knowledge of carbohydrate structures in addition to protein primary structure is a pre-requisite for studying structure/ function relationships in glycoproteins. Primary structu...

Highly sensitive capillary electrophoresis analysis of N‐linked oligosaccharides in glycoproteins following fluorescence derivatization with rhodamine 110 and laser‐induced fluorescence detection

“…Accordingly, we identified the peak around 31.5 min as the triantennary asialo-oligosaccharide with a b1,3-linked galactose. In addition, the relative molar proportions of bi-and triantennary asialo-oligosaccharides are 15 and 85%, which is in good agreement with the values of 17 and 83% that were reported previously [39].…”

Molecular Cloning and Characterization of ConBr, the Lectin of Canavalia Brasiliensis Seeds

“…Sialic acid was determined after sample hydrolysis for 1 h at 80°C with 0.2 M trifluoroacetic acid. Monosaccharides were resolved on a CarboPac PA1 column (0.4 cmX25 cm) eluting at 1 ml/min with either 16 mM NaOH (amino and neutral sugars) or 20 mM NaOH in 60mM sodium acetate and analyzed using a Dionex DX-300 carbohydrate analyzer equipped with a pulsed amperometric detector and the AI-450 chromatographic software (Anumula and Taylor, 1991).…”

Localization and structural characterization of an oligosaccharide O‐linked to bovine PDC‐109 Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa

“…Sialic acid was determined after sample hydrolysis for 1 h at 80°C with 0.2 N trifluoroacetic acid. Monosaccharides were resolved on a CarboPac PA1 column (4 x 250 mm) eluting at 1 ml/min isocratically with either 16 mM NaOH (amino and neutral sugars) or 20 mM NaOH in 60 mM natrium acetate, and analyzed using a Dionex DX-300 carbohydrate analyzer equipped with a pulsed amperometric detector and the AI-450 chromatographic software [16].…”