title: Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.
abstract: Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.
authors.affiliation: Department of Bioengineering, IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
authors.forename: Rimenys J
authors.initials: RJ
authors.lastname: Carvalho
authors.forename: James
authors.initials: J
authors.lastname: Woo
authors.forename: M Raquel
authors.initials: MR
authors.lastname: Aires-Barros
authors.forename: Steven M
authors.initials: SM
authors.lastname: Cramer
authors.forename: Ana M
authors.initials: AM
authors.lastname: Azevedo
mesh_terms: D001897:Boronic Acids / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D012965:Sodium Chloride; D055672:Static Electricity
chemical_list: D001897:Boronic Acids; D006023:Glycoproteins; D012965:Sodium Chloride; C010686:benzeneboronic acid
keywords: Glycoproteins; Lewis bases; Multimodal chromatography; Phenylboronic acid chromatography; cis-Diol groups