Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry

A simple and rapid single‐step method is presented to fabricate an enzyme reactor using trypsin immobilized on a macroporous polymer monolith. A reactor produced in a capillary format 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 trypsin and introduced into the column by capillary force for polymerization/immobilization. The enzyme activity from column‐to‐column is reproducible below 5% relative standard deviation (RSD), while the reactor is durable for at least 20 weeks when stored at room temperature. The apparent kinetic constants V max and K m are of value similar to those obtained by free trypsin in solution. Enzymatic digestion of proteins was shown to be feasible on a time‐scale of seconds and submicromolar concentrations enabling peptide mass mapping by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Copyright © 2004 John Wiley & Sons, Ltd.