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Glycoform Analysis of Alpha1-Acid Glycoprotein by Capillary Electrophoresis Using Electrophoretic Injection

  • Chenhua Zhang
  • William Clarke
  • David S. HageEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1972)

Abstract

Human alpha1-acid glycoprotein (AGP) is an acute phase glycoprotein that has a heterogeneous glycosylation pattern. This pattern can change in certain diseases, which has resulted in interest in using AGP glycoforms as potential biomarkers for these diseases. This report describes a method that uses capillary electrophoresis to characterize and analyze AGP glycoforms both in purified samples of AGP and in human serum. This method uses static and dynamic coatings of poly(ethylene oxide) that are applied to a silica capillary for separation of AGP glycoforms in the reversed-polarity mode of CE and in the presence of negligible electroosmotic flow. Electrophoretic injection is performed onto such capillaries by using a stationary stacking interface between the sample and running buffer. In addition, acidic precipitation and desalting are used to allow for the isolation and the analysis of AGP from only 65 μL of serum. Up to eleven AGP glycoform bands can be reproducibly separated by this method, with the difference in migration time between neighboring bands being 12- to almost 60-fold larger than the standard deviation for the migration time of any given band. A limit of detection down to about 2 nM per glycoform band can be obtained by this method for AGP in serum based on absorbance detection and without the need for further sample modification or labeling.

Key words

Alpha1-acid glycoprotein Capillary electrophoresis Capillary modification Electrophoretic injection Glycoforms Sample stacking Serum 

Notes

Acknowledgement

This work was supported by the National Institutes of Health under grant R01 GM044931.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of NebraskaLincolnUSA
  2. 2.Johns Hopkins University School of MedicineBaltimoreUSA

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