Analysis of glycoproteins

Hydrazinolysis, deamination, and methylation analysis have demonstrated that ai-acid glycoprotein, fetuin, lactotransferrin, and transferrin possess structural features in common.  

Anion-exchange chromatography has been used to separate sugar-borate complexes of D-mannose, L-fucose, D-galactose, D-glucose, D-xylose, L-arabinose, 2-amino-2-deoxy-D-mannose, 2-amino-2-deoxy-D-galactose, l-dcoxy-Ji-arabino-hexose, 2-deoxy-D-/yx u-hexose, and lactose. This technique was used to study the composition of the carbohydrate moieties of ovomucoid, lichenin, and Collocalia mucoid. 

A method for determining the ratio of glycosylated serine to threonine residues in mucins is based on the relative rates of jS-elimination of olefinic amino-acids therefrom. The elimination reaction is faster with glycosylated serine residues. 

on silica gel has been used for the rapid detection of urinary oligosaccharides.  

The lectin from Ricinus communis coupled to Sepharose 4B has been used as an affinity-chromatography matrix in the purification of glycoproteins containing terminal D-galactosyl residues.  

A laser light scattering technique has been used for the measurement of the concentration of glycoproteins when complexed with lectins.A high degree of sensitivity has allowed less than 25 pmol glycoprotein or 4.5 pmol lectin to be detected. 

Methods have been reported for the ultrastructural localization of complex carbohydrates, including glycoproteins, in rat macrophages and monocytes, by employing the reactivity of vicinal glycols to periodate ions and the presence of carboxylate and sulphate ester groups. 

After methanolysis of glycoproteins and 0-trimethylsilylation of the monosaccharides, re-A -acetylation of amino-sugars has been performed directly on gas liquid chromatographic columns, by injecting acetic anhydride together with the sample.Quantitative A -acetylation occurs whilst the O-trimethylsilyl ethers remain intact. 

By using a two column system on a commercially available analyser, amino-acids and the a- and j3-anomers of 2-amino-2-deoxy-D-glucose and 2-amino-2- 

Monosaccharides, derived from polysaccharides, glycolipids, and glycoproteins, have been converted into the corresponding 1-amino-l-deoxy-glycitols by treatment with ammonia and sodium cyanoborohydride, prior to determination using an amino-acid analyser. 

A convenient way to directly analyze glycoproteins by MALDI-MS is to immobilize them on aminophenylboronic acid-derivatized magnetic beads [73], With this approach, isolation of glycoproteins from a complex mixture becomes relatively simple. The protein-immobilized magnetic beads can be spotted directly onto a MALDI sample plate along with a matrix, thereby obviating the need for an additional separation step. 

The latest volume in the series Methods in Carbohydrate Chemistry reports a number of useful techniques, including the proteolytic digestion of membrane-derived glycoproteins, the acid-catalysed hydrolysis and methanolysis of glycoproteins, and the detection of residues linked 0-glycosidically to serine and threonine, Among other methods described are the use of enzymes in the 

A cationic carbocyanine dye ( Stains-all ) has been used to distinguish between sialoglycoproteins, proteins, and lipids following gel electrophoresis in the presence of sodium dodecyl sulphate. Treatment in turn with periodate and dansylhydrazine is claimed to be a very sensitive method for detecting glycoproteins in polyacrylamide gels.  

The monosaccharide content of glycoproteins containing 6—20% carbohydrates has been determined by high voltage electrophoresis of [ H]-labelled sugar alditols. Methods for the carbohydrate analysis of glycoproteins have been reported. Picomolar quantities of monosaccharides in the form of trifluoroacetate derivatives of methyl glycosides are separated and detected by g.l.c. with an electron capture detector. 

A-Acetylneuraminic acid, released from glycoproteins with acid or neuraminidase, has been estimated by g.c.-m.s. of the derived O-trimethylsilyl-ether.  

4-Deoxy-a-L-tAi o-hex-4-enopyranosyluronic acid-containing disaccharides, formed by the action of chondroitin ABC lyase and chondroitin AC lyase have been determined by a periodate-thiobarbituric acid method.  

The 0-trimethylsilyl derivatives of five reference disaccharide alditols, composed of 2-acetamido-2-deoxyhexitol substituted at C-3, C-4, or C-6 with a hexose grouping, have been studied by g.l.c.-m.s. The data were used in the determination of the linkage in disaccharide alditols derived from rat brain glycoproteins and gangliosides. 

Gaieral Aspects, Nomenclature, and Composite Works.—Recommendations of the lUPAC-IUB Commission on Biochemical Nomenclature for the nomenclature of multiple forms of eniymes have been published.  

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Lampio, A. and Finne, J., Sugar analysis of glycoproteins and glycolipids after methanolysis by high-performance liquid chromatography with pulsed amperometric detection, Anal. Biochem., 197, 132, 1991. 

Kakehi, K., Kinoshita, M., and Nakano, M. 2002. Analysis of glycoproteins and the oligosaccharides thereof by high performance capillary electrophoresis - significance in regulatory studies on biopharmaceutical products. Biomedical Chromatography 16(2), 103-115. 

METHYLATION TECHNIQUES IN THE STRUCTURAL ANALYSIS OF GLYCOPROTEINS AND GLYCOLIPIDS... 

Eiljeblad M., Eundblad A., and Pahlsson P. (2002), Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique, Biosens. Bioelectron. 17, 883-891. 

Kakehi, K., and Honda, S. (1996). Analysis of glycoproteins, glycopeptides and glycoprotein-derived oligosaccharides by high-performance capillary electrophoresis.. Chromatogr. A 720, 377-393. 

Kamoda, S., and Kakehi, K. (2006). Capillary electrophoresis for the analysis of glycoprotein pharmaceuticals. Electrophoresis 27, 2495—2504. 

Several model investigations directed toward the analysis of glycoproteins have used methanolysis, and one of the first such model studies was the article by Clamp, Dawson, and Hough86 and their two earlier communications.364,365 These authors examined mixtures containing D-galactose, D-mannose, L-fucose, 2-acetamido-2-deoxy-D-glucose, and neuraminic acid. The solution, after methanolysis, was... 

Analysis of glycoprotein-associated oligosaccharides. Annu. Rev. Biochem. 62, 65-100. 

B. Henrissat A journey through families of carbohydrate-active enzymes Y. Ito Synthesis and functional analysis of glycoprotein glycan chains... 

Amino-4-hydroxybenzoic acid First matrix to be developed specifically for carbohydrates (free glycans) found to be useful for the analysis of glycoprotein A-glycans31... 

G. J. Rademaker and J. Thomas-Oates, Analysis of glycoproteins and glycopeptides using fast atom bombardment, in J. R. Chapman (Ed.), Protein and Peptide Analysis by Mass Spectrometry, Human Press, Totowa, NJ, 1996. 

Additional structural analysis of glycoproteins generally requires fragmentation by chemical or enzymatic cleavage and by separation... 

Cummings, R. D., Merkle, R. A., and Stults, N. L. (1989). Separation and Analysis of Glycoprotein Oligosaccharides. Methods Cell Biol 32 141. 

Jenkins, N., Shah, P. M., and Buckberry, L. D. (2000), Carbohydrate analysis of glycoproteins and glycopeptides, in Reid, R., Ed., Protein and Peptide Drug Analysis, Marcel Dekker, New York. 

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