Incomplete or inconsistent proteolytic digestion is a significant obstacle for quantitative proteomics. High hydrostatic pressure and, specifically, pressure cycling technology (PCT), have been shown to denature proteins, increase protease activity and accelerate protein digestion. Herein we present the results of an investigation of hydrostatic pressure and higher temperature on the specificity and activity of several proteases including trypsin, chymotrypsin, Lys-C and the new enzyme Tryp-N. Heat and pressure cycling can be used to enhance substrate protein denaturation which minimizes reliance on chemical denaturants and boosts recovery of digestion products, while also speeding up the process. Tryp-N, developed at Cold Spring Harbor laboratory and commercialized by Protifi, LLC., is a thermophilic metalloprotease with N-terminal specificity for arginine and lysine. Since basic centers of peptides (the amino terminus and side chains of K/R) occur in one location, b-ions predominate in MS/MS of Tryp-N peptides. Thermal stability of this enzyme prompted us to investigate its performance at high pressure to assess its resistance to pressure denaturation.