Activity of the integrated on-line trypsin microreactor and nanoelectrospray emitter in acetonitrile-water co-solvent mixtures

The on-line trypsin microreactor and nanoelectrospray emitter for peptide mass mapping was demonstrated to be functional under aqueous conditions, but it is well known that electrospray ionization works more efficiently with organic co-solvents. Here, an activity assay was developed to determine the activity of this integrated device with acetonitrile as a co-solvent. Trypsin was immobilized onto fused silica capillaries pulled to fine tips as integrated microreactors coupled as nanoelectrospray ionization emitters. The model substrate N _α-benzoyl-l-Arginine ethyl ester (2.5-20 μM) and an internal standard (N _α-Z-l-Arginine (Z-Arg)) were dissolved in acetonitrile/water at various ratios and infused through the immobilized trypsin microreactor. The trypsin digestion product N _α- benzoyl-l-Arginine (B-Arg) was detected by nanoelectrospray ionization coupled to an ion trap mass spectrometer, and its abundance compared to Z-Arg for quantification. The activity of immobilized trypsin in the microreactor was determined by measuring the ratio of the peak intensities of the hydrolysis product B-Arg to Z-Arg internal standard (three replicates). Kinetic parameters determined from Lineweaver-Burk analysis indicate an enhancement of trypsin activity upon immobilization and the addition of increasing ratios of acetonitrile up to 80 %, where K _m is 0.14 mM and V _max = 1.2 μM/s. Much lower immobilized trypsin activities were noted at 100 % ammonium acetate or 100 % acetonitrile than when the two solvents were mixed. The results clearly indicate that immobilized trypsin retains high biocatalytic activity in 20-80 % acetonitrile and is highly compatible with nanoelectrospray ionization mass spectrometry.