title: Weak anion exchange chromatographic profiling of glycoprotein isoforms on a polymer monolithic capillary.
abstract: High resolution separation of intact glycoproteins, which is essential for many aspects such as finger-print profiling, represents a great challenge because one glycoprotein can exhibit many isoforms with close physicochemical properties. Monolithic columns are important separation media for the separation of intact proteins due to its significant advantages such as easy preparation, high column efficiency and high permeability. However, there are few reports on high resolution profiling of intact glycoproteins. Herein, we presented a polymeric weak anion exchange (WAX) monolithic capillary for high resolution separation of glycoprotein isoforms. A base monolith was first prepared through ring-opening polymerization between tris(2,3-epoxypropyl)isocyanurate and tri(2-aminoethyl), and then modified through reacting with ammonia aqueous solution to convert the unreacted epoxide moieties into primary amino groups. The prepared monolithic capillary was characterized in terms of morphology, pore size, hydrophilicity and reproducibility. The obtained WAX monolithic capillary exhibited desired through-pores and mesopore size, stable skeleton and hydrophilic nature. The performance of the capillary was evaluated using several typical glycoproteins such as α(1)-acid glycoprotein (AGP) as mode analytes. Effects of the experimental parameters on the glycoform resolution were investigated. Under the optimized separation conditions, the tested glycoproteins were all resolved into distinct glycoforms. A comparative investigation with capillary zone electrophoresis (CZE) revealed that this WAX column provided better selectivity as more isoforms were observed, although the resolution of some glycoprotein isoforms decreased.
authors.affiliation: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Lianbing
authors.initials: L
authors.lastname: Ren
authors.forename: Yunchun
authors.initials: Y
authors.lastname: Liu
authors.forename: Hengye
authors.initials: H
authors.lastname: Li
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry*; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D004852:Epoxy Compounds / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D011108:Polymers / Q000737:chemistry; D016062:Porosity; D020033:Protein Isoforms; D015203:Reproducibility of Results; D012965:Sodium Chloride; D014508:Urea / Q000737:chemistry
chemical_list: D000838:Anions; D004852:Epoxy Compounds; D006023:Glycoproteins; D011108:Polymers; D020033:Protein Isoforms; D012965:Sodium Chloride; D014508:Urea