Affinity chromatography with monolithic capillary columnsI. Polymethacrylate monoliths with immobilized mannan for the separation of mannose-binding proteins by capillary electrochromatography and nano-scale liquid chromatography

Abstract

Monolithic capillary columns with surface-immobilized mannan have been introduced for affinity-based micro-column separations by nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). Two kinds of polymethacrylate monoliths were prepared, namely poly(glycidyl methacrylate–co-ethylene dimethacrylate) and poly(glycidyl methacrylate–co-ethylene dimethacrylate–co-[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) to yield neutral and cationic macroporous polymer, respectively. While neutral monoliths with immobilized mannan were only useful for affinity nano-LC, the cationic monoliths with surface-bound mannan were useful in both affinity nano-LC and affinity CEC. The cationic monoliths allowed a relatively high electro-osmotic flow (EOF) when mannan was immobilized to the epoxy monolith via a positively charged spacer arm, triethylenetetramine. The neutral monoliths exhibited lower permeability under pressure-driven flow (PDF) than the cationic monoliths indicating that the latter had wider flow-through pores than the former. Both types of monoliths with immobilized mannan exhibited strong affinity toward mannose-binding proteins (MBP) such as the plant lectins concanavalin A and Lens culinaris agglutinin and a mammalian lectin (e.g. rabbit serum mannose-binding protein). Due to the strong binding affinity, the monoliths with surface bound mannan allowed the injection of large volume of rabbit serum and to isolate in a single run the mannose-binding protein in an amount sufficient to run with it sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), thus demonstrating their capability in “nano-proteomics”.