Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis

Chenhua Zhang, David S. Hage

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

A relatively fast and reproducible CE separation was developed for the glycoform analysis of α1-acid glycoprotein (AGP). Factors that were considered included the pH for this separation and various techniques for coating the capillary and/or to minimize electroosmotic flow and protein adsorption. Optimum resolution of the AGP glycoforms was obtained at pH 4.2 with a running buffer containing 0.1% Brij 35 and by using static and dynamic coatings of PEO on the capillary. These conditions made it possible to separate nine AGP glycoform bands in about 20 min. The limit of detection (based on absorbance measurements) ranged from 0.09 to 0.38 μM for these AGP glycoform bands, and the linear range extended up to a total AGP concentration of at least 240 μM. The migration times for the glycoform bands had typical within-day and day-to-day precisions of ±0.16–0.23% or less, respectively, on a single treated capillary and the variation between capillaries was ±0.56% or less. A charge ladder approach was employed to examine the mass or charge differences in the glycoforms that made up these bands, giving a good fit to a model in which the neighboring bands differed by one charge (e.g., from a sialic acid residue) and had an average mass difference of approximately 0.7–0.9 kDa. The approaches used to develop this separation method are not limited to AGP but could be extended to the analysis of other glycoproteins by CE.

Original languageEnglish (US)
Pages (from-to)102-109
Number of pages8
JournalJournal of Chromatography A
Volume1475
DOIs
StatePublished - Dec 2 2016

Fingerprint

Capillary electrophoresis
Capillary Electrophoresis
Glycoproteins
Acids
Electroosmosis
Coatings
Ladders
N-Acetylneuraminic Acid
Polyethylene oxides
Adsorption
Limit of Detection
Buffers

Keywords

  • Alpha-acid glycoprotein
  • Capillary coating
  • Capillary electrophoresis
  • Charge ladder
  • Glycoform analysis
  • Glycoproteins

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Cite this

Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis. / Zhang, Chenhua; Hage, David S.

In: Journal of Chromatography A, Vol. 1475, 02.12.2016, p. 102-109.

Research output: Contribution to journalArticle

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