The positive effect of Pressure Cycling Technology (PCT) on digestion with trypsin is well established [1-5], and has been shown to result in improved sequence coverage, higher peptide intensities and significantly reduced digestion times. Additionally, the enhancing effect of PCT on the activity of several other enzymes, including Lys-C , chymotrypsin [7, 8], Glu-C , thermolysin , Proteinase K , PNGase F , and lysozyme , has been reported.
Pressure, as well as heat and a variety of chemicals, can be used to denature proteins, but the pressure-perturbed proteins assume conformational forms that are different from those caused by thermal or chemical treatments . Pressure-induced denaturation of substrate proteins leads to better access of enzymes to previously inaccessible, or poorly accessible, target sites. This, in turn, results in improved and accelerated enzyme activity, as long as the level of applied pressure is below the level at which the enzyme itself is denatured and inactivated. Since urea also has a strong denaturing effect on protein conformation, the effect of urea concentration on trypsin digestion under pressure was evaluated. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced trypsin digestion of proteins in urea-containing buffer.