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

Abstract

The on-line trypsin microreactor and nanoelec- trospray 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 emit- ters. The model substrate Na-benzoyl-L-arginine ethyl ester (2.5-20 lM) and an internal standard (Na-Z-L-arginine (Z-Arg)) were dissolved in acetonitrile/water at various ratios and infused through the immobilized trypsin mic- roreactor. The trypsin digestion product Na-benzoyl-L-argi- nine (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 ace- tonitrile up to 80 %, where Km is 0.14 mM and Vmax = 1.2 lM/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.