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 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.

Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

“…Some anionic species such as AGP glycoforms (isoelectric point, 2.8–3.8) [1] can migrate against electroosmotic flow in a bare silica capillary (e.g., when the pH is greater than 3.8 for AGP) [17]. This condition can make it difficult to use electrokinetic injection to apply these anionic solutes onto a capillary in either the normal- or reversed-polarity modes [27].…”

“…New capillaries were activated by rinsing them with 1 M sodium hydroxide for 30 min, followed by a 10 min rinse of water. Modification of the capillaries with static and dynamic coatings of PEO was carried out as described previously [17]. In this method, the capillary was first cleaned by using a 5 min rinse with 1 M sodium hydroxide, followed by a 3 min rinse with water.…”

“…For instance, a relatively fast method for the analysis of AGP glycoforms in aqueous solutions has recently been reported that used both static and dynamic coatings of poly(ethylene oxide) (PEO) in a silica capillary and a pH 4.2 running buffer that contained 0.1% Brij 35. This resulted in a separation of nine glycoform bands within 20 min for standard samples of AGP and with detection limits for the individual glycoform bands that were in the range of 0.09–0.38 μM when using absorbance detection [17]. However, this approach has not yet been adapted for use with more complex biological samples, such as human serum.…”

“…However, this approach has not yet been adapted for use with more complex biological samples, such as human serum. A highly sensitivity method would ideally be needed for such work to analyze samples that contain abnormally low levels of AGP and/or any minor glycoforms that may be present in these samples [1,17]. …”

“…Electrophoretic injection will be performed in the presence of negligible electroosmotic flow by using a capillary with a neutral coating [17], thus providing a stable stacking interface that can provide large loading capacities and long injection times for AGP samples. In addition, both acid precipitation and desalting will be explored as pretreatment methods to allow this CE approach to be used with serum samples.…”