Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

Chen, Fu-Tai A.; Dobashi, Thomas S.; Evangelista, Ramon A.

doi:10.1093/glycob/8.11.1045

Cited by 81 publications

(52 citation statements)

References 11 publications

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“…Other than the capability of direct measuring the formation or the decrease of Neu5Ac in the Neu5Ac synthesis or the Neu5Ac cleavage directions respectively, the CE method has the advantages of high separation efficiency, low sample consumption, and short time of analysis. It is readily applicable for clinical research, food science, and basic research including oligosaccharide, glycoprotein, and polysaccharide analysis (Chen et al 1998;Cheng et al 1998;Hempel 2003;Watanabe and Terabe 2000). The data obtained by the CE method are comparable to those reported previously, confirming the reliability of the method.…”

Section: Discussionsupporting

confidence: 82%

“…The pyruvate formed can also be measured using pyruvate oxidase to produce hydrogen peroxide which is quantified colorimetrically (Sugahara et al 1980). Alternatively, the ManNAc formed can be quantified after fluorescent labeling via reductive amination (Chen et al 1998). The sialic acid formed in the Neu5Ac synthesis direction has been analyzed using periodate-thiobarbituric acid, radioactive, and colorimetric resorcinol-hydrochloric acid methods (Ferrero et al 1996;Pan et al 2004).…”

Section: Reactionmentioning

confidence: 99%

See 1 more Smart Citation

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Cao

et al. 2008

Appl Microbiol Biotechnol

115

151

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Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine (ManNAc). A capillary electrophoresis (CE) assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0-9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg per liter culture) has a higher expression level (2.5 fold) than EcNanA (94 mg per liter culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.

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Section: Discussionsupporting

confidence: 82%

Section: Reactionmentioning

confidence: 99%

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Cao

et al. 2008

Appl Microbiol Biotechnol

115

151

View full text Add to dashboard Cite

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“…The most common derivatization strategy for CE analysis of N-glycans is reductive amination, 45 in which the combination of acidic conditions and high temperatures present a considerable risk for loss of sialic acids, which could contribute to an increased heterogeneity. 46 Removal of the excess of labeling reagent can also be problematic and laborintensive because of the similar properties of the labeling and the derivatized glycans. Therefore, the analysis of underivatized glycans is preferred to reduce the risk of bias because of sample handling and the potential loss of sialic acids.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono-and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins.

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“…Monosaccharide Composition Analysis of BSA Conjugates-Quantitative analysis of the sugar constituents of the sulfated glycoconjugates was performed according to the method developed by Chen et al (12). S4GGnM-BSA and S3GGnM-BSA were hydrolyzed with 2.0 N trifluoroacetic acid to release their constituent sugars.…”

Section: Materials-man/galnac-4-somentioning

confidence: 99%

The Mannose/N-Acetylgalactosamine-4-SO4Receptor Displays Greater Specificity for Multivalent than Monovalent Ligands

Roseman

Baenziger²

2001

Journal of Biological Chemistry

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Recognition of carbohydrates on glycosylated molecules typically requires multivalent interactions with receptors. Monovalent forms of terminal saccharides engaged by the receptor binding sites typically display weak affinities in the mM range and poor specificity. In contrast, multivalent forms of the same saccharides are bound with strong affinity (10 ؊7 -10 ؊9 M) and significantly greater specificity. Although multivalency can readily account for increased affinity, the molecular basis for enhanced specificity is not well understood. We have examined the specificity of the cysteine-rich domain of the mannose/GalNAc-4-SO 4 receptor using monovalent and multivalent forms of the trisaccharide GalNAc␤1,4GlcNAc␤1,2Man␣ (GGnM) sulfated at either the C4 (S4GGnM) or C3 ( We previously demonstrated that glycoproteins such as LH 1 and thyrotropin bearing N-linked oligosaccharides terminating with ␤1,4-linked GalNAc-4-SO 4 are recognized by a receptor located in hepatic endothelial cells, the Man/GalNAc-4-SO 4 receptor, and are rapidly removed from the circulation (1-4). Rapid clearance in conjunction with stimulated release from dense core storage granules in LH-producing cells located in the anterior lobe of the pituitary produce the episodic rise and fall in serum LH levels that is important for the expression of LH bioactivity in vivo. A novel feature of the Man/GalNAc-4-SO 4 receptor is its capacity to bind carbohydrate moieties terminating with Man and GalNAc-4-SO 4 at physically distinct domains that are not structurally related. The binding site for GalNAc-4-SO 4 is located in the cysteine-rich domain found at the N terminus of the Man/GalNAc-4-SO 4 receptor (5). The Cys-rich domain is a member of the ␤-trefoil family of proteins that includes proteins such as acidic fibroblast growth factor. The binding site is a neutral pocket that accommodates the sulfate group, which accounts for the major interactions with the protein (6). Inhibition and modeling studies with monovalent ligands have indicated that the binding site in the Cys-rich domain can also accommodate saccharides with terminal Gal or GalNAc when the sulfate is located at C3 rather than C4 (6, 7).The latter observations were unexpected because we had reported that bovine serum albumin substituted with 6 -8 molecules of SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ (S4GGnM-BSA) is removed from the circulation at a rate that is at least 12-fold greater than that seen for bovine serum albumin substituted with 6 -8 molecules of SO 4 -3-GalNAc␤1,4GlcNAc␤1,2Man␣ (S3GGnM-BSA). Furthermore, isolated hepatic endothelial cells do not bind and internalize S3GGnM-BSA, and the purified Man/GalNAc-4-SO 4 receptor does not display significant binding of S3GGnM-BSA in precipitation assays (2, 3). We have recently determined that the Man/GalNAc-4-SO 4 receptor must be dimeric and engage at least two terminal GalNAc-4-SO 4 moieties on separate oligosaccharides to mediate uptake by hepatic endothelial cells with a K d of 1.63 ϫ 10 Ϫ7 M (8). These observations raise the possibility th...

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Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

Cited by 81 publications

References 11 publications

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

The Mannose/N-Acetylgalactosamine-4-SO4Receptor Displays Greater Specificity for Multivalent than Monovalent Ligands

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