A monolithic PNGase F enzyme microreactor enabling glycan mass mapping of glycoproteins by mass spectrometry

Abstract

We demonstrate a simple and rapid single-step method to fabricate an enzyme microreactor incorporating the N-glycosidase PNGase F (peptide-N-glycosidase F) into a porous polymer-based monolith. The monolith is contained in a capillary format, while the enzyme reactor is ready to use within 1 h of preparation. The monomers making up the monolith, including N-acryloxysuccinimide for covalent immobilization of the enzyme, are mixed with PNGase F and introduced into the column by capillary force for polymerization/immobilization. Glycoproteins (ribonuclease B, asialofetuin, alpha1-acid glycoprotein, and ovalbumin) perfused through the PNGase F reactor were shown to be effectively deglycosylated on a time-scale of seconds/low minutes using low nanogram to microgram per microliter concentration (corresponding to a total sample consumption of 0.1-20 microg of a glycoprotein). The reactor enzyme activity was shown to be reproducible for at least 8 weeks when used and stored at room temperature. Evaluation was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.