Quantification of the α2-6 sialic acid linkage in branched N-glycan structures with capillary nanogel electrophoresis.

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 alpha-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 alpha-1-acid glycoprotein generally containing 0 or 1 α2-6 linked sialic acid.

• Publication year

  2019

• Venue

  Analytical Chemistry

• Publication date

  2019-12-12

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1021/acs.analchem.9b04787PMID 31829566PMCID PMC8631463
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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