Separation Of Oligosaccharides Research Articles

Kinetic characterization of Aspergillus niger chitinase CfcI using a HPAEC-PAD method for native chitin oligosaccharides

The abundant polymer chitin can be degraded by chitinases (EC 3.2.1.14) and β-N-acetyl-hexosaminidases (EC 3.2.1.52) to oligosaccharides and N-acetyl-glucosamine (GlcNAc) monomers. Kinetic characterization of these enzymes requires product quantification by an assay method with a low detection limit, preferably compatible with the use of native, non-labeled substrates. Here we report a quantitative HPAEC-PAD method that allows fast separation of chitin oligosaccharides (COS) ranging from (GlcNac)1–6 at detection limits of 1–3 pmol and a linear range of 5–250 pmol. Quantification under intra- and interday precision conditions was performed with 2.1–5.4% relative standard deviation (RSD) and 1.2–10.3% RSD, respectively. This method was successfully used for the determination of the kinetic parameters of the Aspergillus niger chitinase CfcI with native COS. CfcI was recently shown to release GlcNAc from the reducing end of COS, a new activity for fungal chitinases. A Carbohydrate Binding Module of family 18 (CBM18) is inserted in the CfcI catalytic domain. Site directed mutagenesis was used to assess the functionality of this CfcI-CBM18: four of its key amino acids were replaced by glycine residues, yielding CfcISYNF. Comparison of the kinetic parameters of CfcI and CfcISYNF confirmed that this CBM18 is functionally involved in catalysis.

A dextran-bonded stationary phase for saccharide separation

A-saccharide-based stationary phase for hydrophilic interaction liquid chromatography (HILIC) is presented. The method uses carbonyl di-imidazole (CDI) as a cross-linker for dextran in aqueous solution. Different from the traditional immobilized saccharide stationary phase, this method was using a simple way to bond the high molecular of polysaccharide immobilized on the silica gel. The new method avoided the time-consuming process but had a very considerable loading result. With silica-based surface bonded, it will be possible to develop well-defined surface modifications that promote the hydrophilic with dextran and high mechanical strength with silica. For all tested compounds, including polar compounds and carbohydrates, this dextran-bonded stationary phase performed well in terms of separation efficiency and column stability, and the retention mechanism matched that of typical HILIC retention. Moreover, good selectivity was achieved in the separation of oligosaccharides and glycopeptides.

Chromatographic separation of prebiotic oligosaccharides. Case study: separation of galacto-oligosaccharides on a cation exchanger

Chromatographic separation of prebiotic oligosaccharides, galacto-oligosaccharides from a typical post-reaction mixture containing unreacted substrate lactose and by-products glucose and galactose was investigated. A commercial cation-exchange resin Dowex 50WX4 was chosen as a prospective preparative-scale adsorbent and separation performance of its four ionic forms, H+, Na+, K+ and Ca2+, was tested. Since adsorption equilibrium isotherms were linear within the entire investigated range of concentrations, they were determined by pulse experiments for all saccharides present in a typical post reaction mixture including tri- and tetragalacto-oligosaccharides. From the four counter ions listed above, hydrogen offered the best selectivity and column performance. The selected H+-form of the cation exchanger was further investigated by means of frontal analysis where high ionic strength and elevated viscosity appear and can play a substantial role. Breakthrough curves were measured for monosaccharides, lactose, glucose–lactose mixture and a multicomponent commercial mixture of galacto-oligosaccharides, Vivinal® GOS. The breakthrough curves were successfully described by the dispersive plug flow model with linear driving force approximation.

Characterisation of 3‐aminoquinoline‐derivatised isomeric oligogalacturonic acid by travelling‐wave ion mobility mass spectrometry

Mass spectrometry has become a useful technique for elucidating the chemical structures of oligosaccharides. The combined use of chromatography and mass spectrometry for the separation and identification of oligosaccharides has shown much progress in recent years. However, no powerful method has yet been developed to quickly identify isomeric oligosaccharides in complex mixtures. A rapid travelling-wave ion mobility mass spectrometry (TWIMS-MS) method was developed for the identification of various isomeric oligogalacturonic acids in mixtures and determined their structures, using 3-aminoquinoline (3-AQ) as a labelling agent. TWIMS successfully distinguished isomeric oligogalacturonic acids of various degrees of polymerisation (DPs) and levels of methyl-esterification. After derivatisation by 3-AQ, isomeric oligosaccharides of galacturonic acid, with the DP ranging from 2 to 9 and the number of methyl esters ranging from 1 to 5, were identified by 3-AQ-TWIMS-MS. The isomeric oligosaccharides with varying sites of methyl ester substitution were identified by the post-fragmentation mode of TWIMS using 3-AQ labelling to obtain simplified mass spectra. Using the 3-AQ-TWIMS-MS method, the precise distribution of methyl esters within the pectin molecule and isomeric oligogalacturonic acids after enzyme degradation was determined. Simplified product ion mass spectra and precise analysis of the isomers were achieved by labelling 3-AQ at the reducing end of the oligosaccharides. Series of methyl-esterified galacturonic acid oligomers have predictable drift times, depending on the precise position of the methyl ester.

SAT0415 Diagnostic and predictive value of novel method for analyzing IGG galactosylation in rheumatoid arthritis

BackgroundWhile the emphasis in rheumatoid arthritis (RA) treatment is on early diagnosis and intervention, we cannot provide the certain strategies for diagnosis. It is well known that serum immunoglobulin G...

Ion exchange chromatographic separation and isolation of oligosaccharides of intact low-molecular-weight heparin for the determination of their anticoagulant and anti-inflammatory properties

It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9 × 250mm) ion exchange column with sodium chloride gradient elution and UV detection at 232nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions.

On-line separation and characterization of hyaluronan oligosaccharides derived from radical depolymerization

Hydroxyl radicals are widely implicated in the oxidation of carbohydrates in biological and industrial processes and are often responsible for their structural modification resulting in functional damage. In this study, the radical depolymerization of the polysaccharide hyaluronan was studied in a reaction with hydroxyl radicals generated by Fenton Chemistry. A simple method for isolation and identification of the resulting non-sulfated oligosaccharide products of oxidative depolymerization was established. Hyaluronan oligosaccharides were analyzed using ion-pairing reversed phase high performance liquid chromatography coupled with tandem electrospray mass spectrometry. The sequence of saturated hyaluronan oligosaccharides having even- and odd-numbers of saccharide units, afforded through oxidative depolymerization, were identified. This study represents a simple, effective ‘fingerprinting’ protocol for detecting the damage done to hyaluronan by oxidative radicals. This study should help reveal the potential biological outcome of reactive-oxygen radical-mediated depolymerization of hyaluronan.

Study on preparation and separation of Konjac oligosaccharides

To study the preparation and separation of Konjac oligosaccharides, Konjac Glucomannan was degraded by the combination of γ-irradiation and β-mannanase, and then the degradation product was separated by ultrafiltration. To our interest, for most of Konjac oligosaccharides obtained by this method, the molecular mass was lower than 2200Da. In addition, the 1000Da molecular weight cut off membrane could effectively separate the Konjac oligosaccharides. In conclusion, the combination of γ-irradiation and β-mannanase was an efficient method to obtain Konjac oligosaccharides, and the oligosaccharides of molecular mass lower than 1000Da could be effectively separated by ultrafiltration.

Thermally responsive phospholipid preparations for fluid steering and separation in microfluidics

Aqueous phospholipid preparations comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) are prevalent materials for biological characterization and become gel-like near physiological temperature, but have a low viscosity below 24°C. The rheology of 20% phospholipid preparations of [DMPC]/[DHPC] = 2.5 reveals that, under conditions utilized for fluid steering, the materials are shear-thinning power-law fluids with a power-law index ranging from 0.30 through 0.90. Phospholipid preparations are utilized to steer fluids in microfluidic chips and support hydrodynamic delivery of sample across a double T injection region in a chip. The fact that the phospholipids are fully integrated as a valving material as well as a separation medium is demonstrated through the separation of linear oligosaccharides labeled with 1-aminopyrene-3,6,8-trisulfonic acid.

Influence of molecular configuration and conformation on the electromigration of oligosaccharides in narrow bore capillaries

Capillary electrophoresis enables fast, high efficiency separations of oligosaccharides, wherein positional and/or linkage isomers, bearing the same charge-to-mass ratio, can readily be separated based on hydrodynamic radius differences. Fundamental electrophoretic mobility theory was used to investigate the correlation between changes in hydrodynamic volume equivalent radius and corresponding electrophoretic characteristics of oligosaccharides with different molecular properties. Fluorescently derivatized isomeric malto-, cello-, and isomaltooligosaccharide ladders, differing only in their linkage type of α1→4, β1→4, and α1→6, respectively, as well as a sterically larger N-acetylchitooligosaccharide ladder were used as model compounds. Mere differences in glycosidic linkage type or anomericity of isomeric oligo-glucoses had a decisive impact on their electromigration behavior, thus reflecting discrepancies in hydrodynamic radii and associated molecular conformations. The impact of hydrogen bridges, and associated availability of hydroxyl groups, on the molecular conformations, was investigated by hydrophilic interaction liquid chromatography. The experimentally observed electrophoretic and chromatographic differences between isomeric oligo-glucoses strongly suggested that special attention must be given when homooligosaccharide ladders are employed for normalization and comparability purposes, for example, in glucose unit calculation based structural elucidation.

Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.

Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: Production of well-defined hexasaccharides

Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.

Separation of oligosaccharides from caprine milk whey, prior to prebiotic evaluation

Milk oligosaccharides are believed to have beneficial biological properties. Caprine milk has a relatively high concentration of oligosaccharides in comparison with other ruminant milks and has the oligosaccharide profile closest to that of human milk. The first stage in recovering oligosaccharides from caprine milk whey, a by-product of cheese making, was accomplished by ultrafiltration to remove proteins and fat globules, leaving more than 97% of the initial carbohydrates, mainly lactose, in the permeate. The ultrafiltered permeate was further processed using a 1 kDa ‘tight’ ultrafiltration membrane, which retained less than 7% of the remaining lactose. The final retentate was fractionated by preparative scale molecular size exclusion chromatography to yield 28 fractions; oligosaccharide-rich fractions that were suitable for functionality and gut health promotion testing were detected between fractions 9/10 to 16/17. All fractions were evaluated for their oligosaccharide and carbohydrate profiles using three complementary analytical methods.

Microchip electrophoresis of oligosaccharides using lectin-immobilized preconcentrator gels fabricated by in situ photopolymerization

A lectin-impregnated gel was fabricated at the channel crossing point in a microfluidic chip made from polymethyl methacrylate (PMMA). The acrylamide containing lectin was photopolymerized to form a round gel (radius 60 μm) by irradiation with an argon laser, which was also used for fluorometric detection. This gel was applied to specific concentration, elution, and electrophoretic separation of fluorescent-labeled oligosaccharides. Because the lectin in the polyacrylamide gel was mechanically immobilized, it maintained its activity. The lectin was used to trap up to a few tens of femtomoles of specific oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 2 min by a factor >800, and the amount trapped corresponded to ca. 70% of lectin in the gel. The trapped oligosaccharides were released from the gel by lowering the pH with an acidic background electrolyte. The oligosaccharides that eluted as a broad band were concentrated by transient isotachophoresis stacking using concentrated sodium borate buffer (pH 11.0). The stacked sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, resolution of the saccharides was good, and was similar to that obtained by pinched injection. The method was applied to preconcentration and analysis of oligosaccharides derived from some glycoproteins.

Hydrophilic interaction liquid chromatography coupled to mass spectrometry for the characterization of prebiotic galactooligosaccharides

Three different stationary phases (sulfoalkylbetaine zwitterionic, polyhydroxyethyl aspartamide and ethylene bridge hybrid (BEH) with trifunctionally bonded amide), operating at hydrophilic interaction liquid chromatographic (HILIC) mode, have been assayed and compared for the analysis of complex mixtures of galactooligosaccharides (GOS). Chromatographic methods have been optimized to obtain the best separation between two consecutive galactose containing standards and maltodextrins, measured on the basis of resolution. Influence of several factors such as chemical modifiers (formic acid, ammonium acetate and ammonium hydroxide), organic solvent and gradients of the mobile phases in the separation of oligosaccharides have been studied. The best results were achieved on the BEH amide stationary phase, using acetonitrile:water with 0.1% ammonium hydroxide as mobile phase, where the most of oligosaccharides were successfully resolved. Characteristic MS(2) fragmentation profiles of disaccharides containing galactose, glucose and/or fructose units with different linkages were evaluated and used for the characterization of di-, tri- and tetrasaccharides of three commercial prebiotic GOS mixtures (GOS-1, GOS-2 and GOS-3) by HILIC-MS(n). Similar qualitative and quantitative composition was observed for GOS-1 and GOS-3, whereas different linkages and abundances were detected for GOS-2. In general, (1→4) and (1→6) glycosidic linkages were the main structures found in GOS, although (1→2) and (1→3) linkages were also identified. Regarding molecular weight, up to pentasaccharides were detected in these samples, disaccharides being the most abundant carbohydrates.

Characterizing plant cell wall derived oligosaccharides using hydrophilic interaction chromatography with mass spectrometry detection

Analysis of complex mixtures of plant cell wall derived oligosaccharides is still challenging and multiple analytical techniques are often required for separation and characterization of these mixtures. In this work it is demonstrated that hydrophilic interaction chromatography coupled with evaporative light scattering and mass spectrometry detection (HILIC-ELSD-MS(n)) is a valuable tool for identification of a wide range of neutral and acidic cell wall derived oligosaccharides. The separation potential for acidic oligosaccharides observed with HILIC is much better compared to other existing techniques, like capillary electrophoresis, reversed phase and porous-graphitized carbon chromatography. Important structural information, such as presence of methyl esters and acetyl groups, is retained during analysis. Separation of acidic oligosaccharides with equal charge yet with different degrees of polymerization can be obtained. The efficient coupling of HILIC with ELSD and MS(n)-detection enables characterization and quantification of many different oligosaccharide structures present in complex mixtures. This makes HILIC-ELSD-MS(n) a versatile and powerful additional technique in plant cell wall analysis.

Partial-filling affinity capillary electrophoresis of glycoprotein oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid

Partial-filling affinity capillary electrophoresis has been applied to the simultaneous analysis of interactions between glycoprotein oligosaccharides and certain plant lectins. A lectin solution and a mixture of glycoprotein-derived oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonic acid were introduced to a neutrally coated capillary in this order, and separated by application of a negative voltage. Interaction of a lectin with each oligosaccharide in the mixture was observed as the specific retardation or dissipation of peaks, in addition to the size/charge separation of oligosaccharides by zone electrophoresis in the remainder (≈90%) of the capillary. The strength of the interaction with lectin was controlled by introducing an appropriate volume of lectin solution. Application of various specificities of lectins indicated characteristic migration profiles of the oligosaccharides. Moreover, sequential injection of four lectins ( Maachia amurensis mitogen, Sambucus sieboldiana agglutinin, Erythrina cristagalli agglutinin, Aleuria aurantia lectin) induced complete dissipation of complex-type oligosaccharides and enabled specific determination of the presence of high-mannose oligosaccharides without the interference or alteration of the electropherogram in porcine thyroglobulin. This method was also applied to determine the binding constants of ovalbumin-derived oligosaccharides to wheat germ agglutinin.

Evaluation of different operation modes of high performance liquid chromatography for the analysis of complex mixtures of neutral oligosaccharides

Chromatographic methods based on different HPLC operation modes, reverse phase (RP), high performance anion exchange chromatography (HPAEC), graphitized carbon chromatography (GCC) and hydrophilic interaction liquid chromatography (HILIC), have been developed and compared for the analysis of complex mixtures of neutral oligosaccharides with functional properties. Whereas GCC gave the best chromatographic separation of isomeric oligosaccharides with the same molecular weight ( R s values in the range 1.0–4.0 and 2.4–5.6 for tetra- and pentasaccharides, respectively), HILIC provided the best results for mixtures including oligosaccharides of different degrees of polymerization ( R s values of maltooligosaccharides between 3.4 and 6.2). Validation of the HILIC LC–MS method proved its utility for the analysis of oligosaccharide mixtures with functional properties: relative standard deviations lower than 10%, LOD's and LOQ's in the range 12.7–130.2 ng mL −1 and 39.3–402.2 ng mL −1, respectively, and linearity up to 10–20 μg mL −1. Quantitative data for fructooligosaccharides, gentiooligosaccharides and dextransucrase cellobiose acceptor oligosaccharides were obtained by using this method.

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.

A novel zwitterionic HILIC stationary phase based on “thiol-ene” click chemistry between cysteine and vinyl silica

A novel zwitterionic stationary phase with high hydrophilicity was facilely synthesized based on the "thiol-ene" click reaction between cysteine and vinyl silica, which exhibited great potential in the separation of oligosaccharides, peptides and basic compounds, as well as in the enrichment of glycopeptides.