Dataset Information

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of ?(1-3)-linked galactose in glycans.

ABSTRACT: A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary. The nanogel is compatible with enzymes and lectins and improves the stability of galactosidase, enabling more cost-effective use of biological reagents that provide insight into glycan structure. A feature of using stationary zones is that the reaction time can be controlled by the length of the zone, the applied field controlling the analyte mobility, or the use of electrophoretic mixing by switching the polarity of the applied voltage while the analyte is located in the zone. The temperature, applied voltage, and length of the stationary zone, which are factors that enhance the performance of the enzyme, are characterized. The combined use of enzymes and lectins in capillary electrophoresis is a new strategy to advance rapid and automated analyses of glycans using nanoliter volumes of enzymes and lectins. The applicability of this use of stationary zones of enzyme and lectin in capillary electrophoresis is demonstrated with the identification of ?(1-3)-linked galactose in N-glycan.

SUBMITTER: Holland LA

PROVIDER: S-EPMC5675761 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Publications

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1-3)-linked galactose in glycans.

Holland Lisa A LA Gattu Srikanth S Crihfield Cassandra L CL Bwanali Lloyd L

Journal of chromatography. A 20170616

A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary. The nanogel is compatible with enzymes and lectins and improves the stability of galactosidase, enabling more cost-ef ...[more]

PMID: 28647147

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Project description:Glycosylated human IgG contains fucosylated biantennary N-glycans with different modifications including N-acetylglucosamine, which bisects the mannose core. Although only a limited number of IgG N-glycan structures are possible, human IgG N-glycans are predominantly biantennary and fucosylated and contain varying levels of ?2-6-linked sialic acid, galactose, and bisected N-acetylglucosamine. Monitoring the relative abundance of bisecting N-acetylglucosamine is relevant to physiological processes. A rapid, inexpensive, and automated method is used to successfully profile N-linked IgG glycans and is suitable to distinguish differences in bisection, galactosylation, and sialylation in N-glycans derived from different sources of human IgG. The separation is facilitated with self-assembled nanogels that also contain a single stationary zone of lectin. When the lectin specificity matches the N-glycan, the peak disappears from the electropherogram, identifying the N-glycan structure. The nanogel electrophoresis generates separation efficiencies of 500?000 plates and resolves the positional isomers of monogalactosylated biantennary N-glycan and the monogalactosylated bisected N-glycan. Aleuria aurantia lectin, Erythrina cristagalli lectin (ECL), Sambucus nigra lectin, and Phaseolus vulgaris Erythroagglutinin (PHA-E) are used to identify fucose, galactose, ?2-6-linked sialic acid, and bisected N-acetylglucosamine, respectively. Although PHA-E lectin has a strong binding affinity for bisected N-glycans that also contain a terminal galactose on the ?1-6-linked mannose branch, this lectin has lower affinity for N-glycans containing terminal galactose and for agalactosylated bisected biantennary N-glycans. The lower affinity to these motifs is observed in the electropherograms as a change in peak width, which when used in conjunction with the results from the ECL lectin authenticates the composition of the agalactosylated bisected biantennary N-glycan. For runs performed at 17 °C, the precision in migration time and peak area was less than or equal to 0.08 and 4% relative standard deviation, respectively. The method is compatible with electrokinetic and hydrodynamic injections, with detection limits of 70 and 300 pM, respectively.

Dataset Information

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of ?(1-3)-linked galactose in glycans.

Publications

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1-3)-linked galactose in glycans.

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