Chapter 5 Analysis of Glycoconjugates Using High-pH Anion-Exchange Chromatography

Townsend, R. Reid

doi:10.1016/s0301-4770(08)60510-2

Cited by 28 publications

(5 citation statements)

References 127 publications

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“…Neutral and amino monosaccharides were released by hydrolysis with 100 µl of 2 M HCl for 2 h at 100 mC, and sialic acids were released from the protein with 100 µl of 0.1 M HCl for 1 h at 70 mC. The samples were dried and washed three times with 100 µl of water prior to analysis by high-pH anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) [27]. HPAEC-PAD of neutral and amino monosaccharides was carried out using a CarboPac PA1 column (4 mmi250 mm ; Dionex, Camberley, Surrey, U.K.) fitted with amino trap [28] and borate trap guard columns [29].…”

Section: Monosaccharide Analysismentioning

confidence: 99%

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen

Shaw

Woods

Myers

et al. 2002

Biochemical Journal

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The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.

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Section: Monosaccharide Analysismentioning

confidence: 99%

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen

Shaw

Woods

Myers

et al. 2002

Biochemical Journal

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“…In contrast to the reported observations that AA is not suitable for analysis using ion-exchange, reversed phase and mass spectrometry (Bigge et al,1995), we have found that indeed AA is the preferred label over the AB for carbohydrates and their characterization using variety of biochemical and spectroscopic techniques. In addition, it is being proposed that all the applications developed for characterization of carbohydrates using HPAEC-PAD (for review see Townsend, 1995) and HPLC methods using 2-aminopyridine (Hase, 1994) are directly applicable to the versatile AA labeled oligosaccharides, including the analysis by gel electrophoresis (Bigge et al, 1995). Obviously, current methods available for analysis of the reduced oligosaccharides are not directly applicable to this technology.…”

Section: Introductionmentioning

confidence: 99%

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

1998

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Facile labeling of oligosaccharides (acidic and neutral) in a nonselective manner was achieved with highly fluorescent anthranilic acid (AA, 2-aminobenzoic acid) (more than twice the intensity of 2-aminobenzamide, AB) for specific detection at very high sensitivity. Quantitative labeling in acetate-borate buffered methanol (approximately pH 5.0) at 80 degreesC for 60 min resulted in negligible or no desialylation of the oligosaccharides. A high resolution high performance liquid chromatographic method was developed for quantitative oligosaccharide mapping on a polymeric-NH2bonded (Astec) column operating under normal phase and anion exchange (NP-HPAEC) conditions. For isolation of oligosaccharides from the map by simple evaporation, the chromatographic conditions developed use volatile acetic acid-triethylamine buffer (approximately pH 4.0) systems. The mapping and characterization technology was developed using well characterized standard glycoproteins. The fluorescent oligosaccharide maps were similar to the maps obtained by the high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), except that the fluorescent maps contained more defined peaks. In the map, the oligosaccharides separated into groups based on charge, size, linkage, and overall structure in a manner similar to HPAEC-PAD with contribution of -COOH function from the label, anthranilic acid. However, selectivity of the column for sialic acid linkages was different. A second dimension normal phase HPLC (NP-HPLC) method was developed on an amide column (TSK Gel amide-80) for separation of the AA labeled neutral complex type and isomeric structures of high mannose type oligosaccharides. The oligosaccharides labeled with AA are compatible with biochemical and biophysical techniques, and use of matrix assisted laser desorption mass spectrometry for rapid determination of oligosaccharide mass map of glycoproteins is demonstrated. High resolution of NP-HPAEC and NP-HPLC methods combined with mass spectrometry (MALDI-TOF) can provide an effective technology for analyzing a wide repertoire of oligosaccharide structures and for determining the action of both transferases and glycosidases.

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“…HPAEC-PAD is widely employed in the analysis of oligosaccharides and there are many studies. Oligosaccharides digested by enzymolysis or hydrolysis can be monitored using this technique, such as the preparation of pectic oligosaccharides [55,[95][96][97][98][99], fungal oligosaccharide [100,101], low-molecular-weight heparins [102], agarooligosaccharides [103] and glycoprotein oligosaccharides [104,105], etc. For example, Zheng and colleagues utilized HPAEC-PAD to evaluate the hydrolysates of galactocan with different partial acid hydrolysis times.…”

Section: High-performance Anion Exchange Chromatographymentioning

confidence: 99%

Application of chromatography in purification and structural analysis of natural polysaccharides: A review

Zheng

Yan

Cao

et al. 2023

J of Separation Science

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Polysaccharides are widely distributed in natural sources from monocytic microorganisms to higher animals, and are found in a variety of biological activities in recent decades. Natural polysaccharides have the characteristics of large molecular weight, diverse composition, and complex structure, so their purification and structural analysis are difficult issues in research. Chromatography as a powerful separation technique, plays an irreplaceable role in the separation and structural analysis of natural polysaccharides, especially in the purification of polysaccharides, the separation of hydrolysates, and the analysis of monosaccharide composition. The separation mechanisms and application of different chromatographic methods in the studies of polysaccharides were summarized in this review. Moreover, the advantages and drawbacks of various chromatography methods were discussed as well.

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Chapter 5 Analysis of Glycoconjugates Using High-pH Anion-Exchange Chromatography

Cited by 28 publications

References 127 publications

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen

High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Application of chromatography in purification and structural analysis of natural polysaccharides: A review

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