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.