Critical assessment of the spectroscopic activity assay for monitoring trypsin activity in organic–aqueous solvent

Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous–organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems. However, as reported here, the changing solvent polarity induces bias in the absorbance measurement, lending upward of 35% error in the apparent trypsin activity as the acetonitrile is raised to 70%. Furthermore, time-dependent spectroscopic and mass spectrometric measurements reveal a progressive deactivation of trypsin over a 5- to 10-min period in as little as 30% acetonitrile.