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

• Published in: European journal of biochemistry Vol. 195; no. 1; pp. 269 - 280
• Main Authors: ANUMULA, Kalyan Rao, TAYLOR, Paul B.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.01.1991; Blackwell
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Asparagine; Biological and medical sciences; Carbohydrate Conformation; Carbohydrate Sequence; Chromatography, Ion Exchange; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - chemistry; Glycoside Hydrolases; Humans; Indicators and Reagents; Methylation; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - isolation & purification; Proteins; Trypsin; Enzymatic digestion; Chemical composition; Glycoproteins; Investigation method; Oligosaccharide; Ion exchange chromatography
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1991.tb15703.x

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 N‐glycosidase F, endo‐β‐N‐acetylglucosaminidase F and endo‐β‐N‐acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion‐exchange 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 μA 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 α1‐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 α‐mannosidase. Treatment with β‐galactosidase, βN‐acetylglucosaminidase and α‐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 α‐l‐fucosidase, endo‐β‐N‐acetylglucosaminidase F and endo‐β‐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 endo‐and 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.