Monosaccharides In Glycoproteins Research Articles

Sensitive determination of rhodamine 110-labeled monosaccharides in glycoprotein by capillary electrophoresis with laser-induced fluorescence detection

Rhodamine 110 (Rho110) has been used in the highly sensitive analysis of monosaccharides, as it reacts with the reducing carbonyl group of the saccharides. The monosaccharide derivatives were investigated by capillary electrophoresis with laser-induced fluorescence detection. The derivatization was performed at 90 degrees C for 30 min for all monosaccharides. The derivatized monosaccharides were separated using 200 mM borate (pH 10.5) as running buffer within 20 min. The fluorescence intensities of Rho110-derivatives were significantly decreased by the presence of excess reducing agent, but were greatly increased by the addition of potassium hexacyanoferrate(III). The concentration and mass detection limits for monosaccharides were in the range of 1.4-2.8 nM and 36-70 amol, respectively. We have applied this derivatization method to the analysis of the composition of monosaccharides in glycoproteins (ribonuclease B, fetuin, and erythropoietin) following their subjection to strong acid hydrolysis. The results from these analyses were in good agreements with the reported values established previously.

Multiple-Reaction Monitoring Liquid Chromatography Mass Spectrometry for Monosaccharide Compositional Analysis of Glycoproteins

A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M - H](-). The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 microg of fetuin, ribonuclease B, peroxidase, and alpha(1)-acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. It does not require derivatization or postcolumn addition of reagents.

CarboPac™ PA20: a new monosaccharide separator column with electrochemical detection with disposable gold electrodes

This report documents the development of a new monosaccharide separator column (CarboPac PA20, 3×150 mm) that allows fast, efficient monosaccharide separations with good spacing. It is based on a new chemistry with a reduced resin particle size (from 10 to 6.5 μm). Faster, more efficient separations of glycoprotein monosaccharides with better spacing were achieved across a range of isocratic NaOH concentrations at lower flow rates. Detection sensitivity was improved, enabling routine low to sub pmol monosaccharide determinations. Glycoprotein monosaccharides eluted in less than 10 min at a flow rate of 0.5 ml/min. Furthermore, when used with an AminoTrap guard column, the protein matrix consisting of amino acids and peptides (released by acid hydrolysis of glycoprotein) did not interfere with monosaccharide analysis. Compared to previous CarboPac columns (CarboPac PA1 and CarboPac PA10), the CarboPac PA20 has improved selectivity with respect to glycoprotein monosaccharides. The improved selectivity results in better separation of glucosamine and galactose, enabling the accurate determination of monosaccharide ratios for undergalactosylated glycoproteins. Finally, disposable gold working electrodes that eliminate the possibility of working electrode recession affecting peak area response were used.

Reversed-Phase HPLC of Monosaccharides in Glycoproteins Derivatized with Aminopyrazine with Fluorescence Detection

A precise method for the determination of monosaccharides is described. Monosaccharides were separated as their aminopyrazine derivatives using reversed-phase HPLC and were detected at 410 nm by a fluorescence detector with excitation at 245 nm. The 8 monosaccharides tested were completely separated in about 20 min, and the detection limits were 45-800 fmol. Particularly, strong fluorescence was observed for N-acetylated monosaccharide and sensitive determination could be carried out. The monosaccharide composition of several glycoproteins was also determined by reversed-phase HPLC and anion-exchange HPLC, and the results were consistent with the reported values.

Quantitative Cleavage of theN-Glycosidic Bond under the Normal Conditions of Methanolysis Used for the Analysis of Glycoprotein Monosaccharides

The most common method used for the liberation of monosaccharides from glycoproteinN-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides asO-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively theN-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave theN-glycosidic bond (96%), liberating glucosamine (and not itsO-methylglycosides) and other minor reaction products which were identified. Because otherN-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as theO-methylglycosides of glucosamine, the GlcNAc residue involved in theN-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.

Sensitive Determination of Glycoprotein Monosaccharides by Gas Chromatography with Electron Capture Detection

Sensitive Determination of Glycoprotein Monosaccharides by Gas Chromatography with Electron Capture Detection

Separation of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides by capillary electrochromatography.

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of component monosaccharides in glycoproteins (fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine) and epimeric aldopentoses (arabinose, lyxose, ribose and xylose) were well separated from each other by capillary electrochromatography on a Hypersil ODS column with a mixture of 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer, pH 6.0 to approximately 6.3, and acetonitrile (2.2:1 v/v) as eluent. The elution of these compounds showed relatively strong dependence on the pH and concentration of the buffer salts contained in the eluent, as compared to the elution by pressure-driven high-performance liquid chromatography (HPLC) on the same stationary phase, but separation of PMP-monosaccharides was better than that by HPLC. Retention times of PMP-monosaccharides were highly reproducible with a relative standard deviation (RSD) of approximately 0.6%, and quantification with an RSD less than 5% could be achieved using 3-O-methylglucose as an internal standard.

Simultaneous Determination of Monosaccharides in Glycoproteins by Capillary Electrophoresis

A rapid, easy, and reproducible capillary electrophoretic method for the simultaneous determination of acidic, neutral, and amino sugars and sugar alcohols was developed. Underivatized mannuronic acid, glucuronic acid, galacturonic acid,N-glycolylneuraminic acid,N-acetylneuraminic acid, glucosamine, galactosamine, mannose, xylose, glucose, galactose, fucose, ribose, mannitol, sorbitol, xylitol, and inositol were simultaneously determined with indirect UV detection using 2,6-pyridinedicarboxylic acid as a background electrolyte. A highly alkaline pH condition was used in order to charge carbohydrates negatively and to promote migration toward the anode. Electroosmotic flow was reversed to the direction of the anode by adding cetyltrimethylammonium bromide to the electrolyte. The separation of the carbohydrates was investigated by optimizing the operating pH value and satisfactory resolution was obtained at pH 12.1. The relative standard deviations of the method for carbohydrates were between 0.02 and 0.33% for migration times and were greater than 2.7% for peak areas (n= 6). The minimum detectable level ranged from 23 to 71 μM with a 6-nl injection at a signal-to-noise ratio of 3. This method was applied to the composition analysis of monosaccharides in glycoprotein. After acid hydrolysis of fetuin under each optimum condition, sialo, neutral, and amino sugars were quantified under the same electrophoretic condition.

A strategy for chromatographic and structural analysis of monosaccharide species from glycoproteins.

A general strategy for the chromatographic and structural analysis of the monosaccharide species fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), glucose (Glc), mannose (Man), N-acetylneuraminic acid (NANA) present in glycoproteins is described. Qualitative and quantitative aspects for the separation of these glycoprotein monosaccharides (monosaccharide species) using ligand-exchange chromatography (LEC) and high pH anion-exchange chromatography (HPAEC) in combination with pulsed-amperometric detection (PAD), refractive index (RI) and ultraviolet (UV) monitoring are discussed in detail. The conditions for the acidic hydrolysis of glycoproteins and for the liquid chromatographic analyses of glycoprotein monosaccharides using HPAEC and LEC technique were optimised. Furthermore, the characterisation of glycoproteins according to their purity and molecular mass connected with a comparison to biomolecules that are not glycosylated or whose extent of glycosylation is low was carried out by means of matrix-assisted laser-desorption ionisation mass spectrometry (MALDI-MS). The identification of glycoprotein monosaccharides using an on-line coupling liquid chromatography mass spectrometry (LC-MS/MS) was performed by means of their characteristic "quasi molecule ions" such as (M + NH(4))(+) and (2M + NH(4))(+). The different chromatographic and structural methods used in combination with each other were applied to characterise and determine the monosaccharide species of fetuin and a membrane glycoprotein fraction.

Quantitative Determination of Monosaccharides in Glycoproteins by High-Performance Liquid Chromatography with Highly Sensitive Fluorescence Detection

For specific determination of monosaccharides with high sensitivity, glycoprotein acid hydrolysates were derivatized in a simple step with excess anthranilic acid (2-aminobenzoic acid) in the presence of sodium cyanoborohydride to give highly fluorescent stable derivatives. The monosaccharide derivatives were completely separated from the excess reagent and from each other by HPLC on a C-18 reversed-phase column using a 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase. Reductive amination of the monosaccharides in the methanol-acetate-borate medium was complete within 20 min at 80°C. Derivatization of glucosamine with the anthranilic acid was accompanied by epimerization to mannosamine (>15%) in methanol-acetic acid reaction medium, but it was reduced to <3% in methanol-acetate-borate reaction medium. Fluorescence intensity of the hexosamines was greater than twice the intensity of the neutral monosaccharides. The fluorescent derivatives had excitation maxima at 230, 245, and 360 nm and an emission maximum at 425 nm. Fluorescence intensity at 230 nm excitation was about 10 times greater than that obtained with excitation at 360 nm for all the monosaccharides. Release and concomitant destruction of the monosaccharides during hydrolysis in 20% TFA at 100°C for 7-8 h resulted in 83-85% recovery of all the monosaccharides from glycoproteins. The monosaccharide compositions determined by this method were in excellent agreement with the expected values for a recombinant immunoglobulin and fetuin and were highly reproducible. Relative standard deviation for the composition determinations and precision was less than 3%. Because of the high sensitivity of this method (∼100 fmol using a analytical column), it is suitable for analyzing less than 1.0 μg of glycoprotein.

Determination of Monosaccharides in Glycoproteins by Reverse-Phase High-Performance Liquid Chromatography

This report examines monosaccharide composition and content using the reverse-phase high-performance liquid chromatographic separation of p-aminobenzoic ethyl ester derivatives of neutral and amino sugars released from glycoproteins. After acid hydrolysis of glycoproteins with 2 M trifluoroacetic acid, sugars are derivatized with p-aminobenzoic ethyl ester (ABEE), which strongly absorbs uv light at 254 nm in the presence of sodium cyanoborohydride. Reverse-phase (RP)HPLC of the ABEE-sugar derivatives is performed on Pico · Tag column in an isocratic mode. RP-HPLC conditions are optimized by using ternary mixture as a mobile phase and 45°C as a column temperature. This procedure is very useful for the simultaneous analysis of neutral and amino sugars in a single chromatographic step using RP-HPLC without reacetylation of deacetylated amino sugars, which were produced by acid hydrolysis, and additional chromatography to remove side products. Monosaccharide composition and contents determined on glycoproteins by this method were very comparable to those previously obtained by other techniques.

Determination of monosaccharides in glycoproteins by reversed-phase high-performance liquid chromatography on 2.1-mm narrowbore columns

The monosaccharide composition of glycoproteins was determined by using a Supelcosil LC-18 150 × 2.1 mm narrowbore column under reversed-phase conditions. Methodology was developed for sample hydrolysis, N-acetylation, benzoylation, and cleanup prior to high-performance liquid chromatography. Monosaccharides were identified and quantified at picomole levels. Various glycoproteins were then analyzed, including ribonucleases A and B, Mucin, and fetuin. In addition, the monosaccharide content of Chinese hamster ovary cell-expressed recombinant human renin was determined.

Glycosylation of proteins in developing human brain.

The carbohydrate content of human brain glycoproteins was studied during development from the age of 12 fetal weeks to 8 postnatal months. The concentration of all the glycoprotein monosaccharides increased with age. The total amount of glycoprotein monosaccharides per lipid-free dry tissue increased by about 150% from the end of the third fetal month to the time of delivery. The increase leveled off around term, and only minor increase occurred after birth. The adult level was reached by the fifth postnatal month.

Microdetermination of monosaccharides in glycoproteins by gas-liquid chromatography

In a newly developed method for determining picomolar quantities of monosaccharides in glycoproteins, the methyl glycosides released by methanolysis of glycoproteins are separated as trifluoroacetates by gas-liquid chromatography and are detected with an electron capture detector. The application of the method has been demonstrated for the analysis of the carbohydrate moiety of secretory component isolated from human colostral immunoglobulin A.

Microdetermination of monosaccharides in glycoproteins: I. Electrophoretic methods

A sensitive method that has been developed for determination of monosaccharides in glycoproteins is based on high-voltage electrophoretic separation of tritium-labeled monosaccharides. Precise quantitation is mediated by using internal standards, specific for each monosaccharide in the sample. The application of this method and its reproducibility has been demonstrated by analysis of the carbohydrate moiety of immunoglobulins.

On the automated analysis of neutral monosaccharides in glycoproteins and polysaccharides

An improved method for the automated, quantitative analysis of glycoproteins and polysaccharides for neutral monosaccharide components has been developed, based on the ion-exchange chromatography at pH 7 of sugar-borate complexes. The destruction of sugars during acid hydrolysis has been investigated, and a variety of methods for the neutralization of hydrolysates have been evaluated.

Determination of neutral sugars in glycoproteins by gas—liquid chromatography

A procedure is described for the quantitative hydrolysis, derivatization, and analysis by gas—liquid chromatography of the neutral monosaccharides in glyco-proteins. The new liquid phase combination employed gives resolution of eleven alditol acetates and shows a low level of baseline drift during temperature programming. Application of the analysis to a number of different glycoproteins is given.