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

Abstract

A method based on capillary electrophoresis (CE) with electrophoretic injection and absorbance detection was developed for the direct analysis of AGP glycoforms in human serum. Electrophoretic injection of AGP was performed in the reversed-polarity mode of CE with a capillary coated with poly(ethylene oxide) and that had minimal electroosmotic flow. This situation created an essentially stationary interface between the sample and running buffer during injection and sample stacking. This approach allowed an 11,000-fold increase in sample loading for a 5 min injection versus hydrodynamic injection and without introducing any significant levels of extra band-broadening. This method was used with sample pretreatment methods based on acid precipitation and desalting to examine AGP glycoforms in only 65 μL of serum. A limit of detection of 2.1–11.3 nM was obtained for the major AGP glycoform bands in serum, and the sample pretreatment method gave a recovery of 72.3–80.9% for these glycoforms. The precision for the migration times was ± 0.08–0.13% and the precision for the peak areas was ± 0.34–1.18% when using serum samples and an internal standard. This method was used for both normal pooled serum and serum from individuals with systemic lupus erythematosus. Results were obtained in a separation time of 25 min and allowed the comparison of up to eleven glycoform bands in these samples. A similar approach may be useful in examining additional glycoproteins in serum or other types of biological samples.

Recent progress of sample stacking in capillary electrophoresis (2016–2018)

“…Zhang et al. described a method for direct analysis of alpha 1 ‐acid glycoprotein glycoforms in human serum. Electrophoretic injection was performed in the reversed‐polarity mode with a poly(ethylene oxide) coated capillary with minimum EOF.…”

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016–2018)

“…FASS and FASI are usually the first choice among all stacking techniques due to their simplicity and high compatibility with different CE modes such as CZE , EKC , CGE , CEC as well as microchip electrophoresis . An uncomplicated buffer system used in FASS and FASI allows to utilize a whole range of detectors including UV , LED‐IF , LIF , C4D , and amperometric .…”

“…FASS and FASI are usually the first choice among all stacking techniques due to their simplicity and high compatibility with different CE modes such as CZE , EKC , CGE , CEC as well as microchip electrophoresis . An uncomplicated buffer system used in FASS and FASI allows to utilize a whole range of detectors including UV , LED‐IF , LIF , C4D , and amperometric . The sample matrix effect is reduced or eliminated since the sample clean‐up is necessary to lower the conductivity of the sample and thus, FASS and FASI can be used in analysis of biological , pharmaceutical , environmental , food , industrial , and forensic samples .…”

“…An uncomplicated buffer system used in FASS and FASI allows to utilize a whole range of detectors including UV , LED‐IF , LIF , C4D , and amperometric . The sample matrix effect is reduced or eliminated since the sample clean‐up is necessary to lower the conductivity of the sample and thus, FASS and FASI can be used in analysis of biological , pharmaceutical , environmental , food , industrial , and forensic samples . In addition, combination of FASS and FASI with other stacking techniques is popularly used in multi‐stacking approaches which will be discussed further in this review.…”

Nanoparticle-supported polymer brushes for temperature-regulated glycoprotein separation: investigation of structure–function relationship

“…based on capillary electrophoresis (CE) also use complicated protocols and require specialized instruments. 22 Among all the methods used for glycoprotein enrichment, boronic acid-based affinity separation has become increasingly popular because of its low bias, long shelf time and convenient handling. [23][24][25][26][27][28] In particular, polymer materials containing boronate affinity ligands can be designed to offer selective glycoprotein enrichment and separation, as boronic acids can form stable ester bonds with cis-diol groups at the carbohydrate site of glycoproteins.…”