Separation of Glycoproteins Based on Sugar Chains Using Novel Stationary Phases Modified with Poly(ethylene glycol)-Conjugated Boronic-Acid Derivatives

Boronic acid (BA) reversibly complexes with the diol structure. BA derivatives separate glycoproteins based on the differences in the sugar chains. Separation typically occurs under basic conditions, which does not guarantee the structural stability of glycoproteins. Here, 5-boronopicolinic acid (BPA) is used to prepare silica-gel based columns with poly(ethylene glycol) (PEG) as a linker to suppress nonselective adsorption and poly(ethylene imine) (PEI) as a scaffold to increase the BPA moiety density. Highperformance liquid chromatography (HPLC) using only aqueous buffer solutions without organic solvents demonstrates the selective retention ability of the BPA columns for glycoproteins. BPA interacts with the diols in the sugar chains but not the proteins. In an evaluation for N-glycans, the BPA columns show a higher retention ability toward high mannose type and a lower affinity to N-acetylneuraminic acid (Neu5Ac). Finally, a pair of glycoproteins, fetuin and asialofetuin, are selectively separated due to the presence of Neu5Ac on the nonreducing end.

Automated summary

The study focuses on the selective separation of glycoproteins using BPA columns. These columns have a high retention ability for high mannose type and a low affinity for N-acetylneuraminic acid.