Dataset Information

Quantification of the α2-6 Sialic Acid Linkage in Branched N-Glycan Structures with Capillary Nanogel Electrophoresis.

ABSTRACT: Sialylation and sialic acid linkage in N-glycans are markers of disease but are analytically challenging to quantify. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated N-glycans in real time in the capillary so that N-glycan structures containing α2-6-linked sialic acid are easily separated, detected, and quantified. In this study, N-glycans were sequentially cleaved by enzymes at the head of the separation capillary so that the presence of α2-6-linked sialic acids corresponded to a shift in the analyte migration time in a manner that enabled interpretation of the N-glycan structure. Following injection, only afucosylated N-glycan structures were passed through enzyme zones that contained α2-3 sialidase, followed by β1-3,4 galactosidase, which cleaved any terminal α2-3-linked sialic acid and underlying galactose yielding a terminal N-acetyl glucosamine. With this treatment complete, a third zone of α2-3,6,8 sialidase converted the remaining α2-6-linked sialic acid to terminal galactose. With these enzyme processing steps the α2-6-linked sialic acid residues on an N-glycan correlated directly to the number of terminal galactose residues that remained. The number of terminal galactose residues could be interpreted as a stepwise decrease in the migration time. Complex N-glycans from α-1-acid glycoprotein were analyzed using this approach, revealing that a limited number of α2-6-linked sialic acids were present with biantennary, triantennary, and tetraantennary N-glycans of α-1-acid glycoprotein generally containing 0 or 1 α2-6-linked sialic acid.

SUBMITTER: Bwanali L

PROVIDER: S-EPMC8631463 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

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

Quantification of the α2-6 Sialic Acid Linkage in Branched N-Glycan Structures with Capillary Nanogel Electrophoresis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets