title: Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis.
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 20min. The limit of detection (based on absorbance measurements) ranged from 0.09 to 0.38μM for these AGP glycoform bands, and the linear range extended up to a total AGP concentration of at least 240μM. 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 0.7-0.9kDa. 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.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
authors.forename: Chenhua
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA. Electronic address: dhage1@unl.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D002021:Buffers; D053841:Electroosmosis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009961:Orosomucoid / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D002021:Buffers; D009961:Orosomucoid; D020033:Protein Isoforms; D019158:N-Acetylneuraminic Acid
keywords: Alpha(1)-acid glycoprotein; Capillary coating; Capillary electrophoresis; Charge ladder; Glycoform analysis; Glycoproteins