Introduction

The rate, specificity, and efficiency of proteolytic digestion profoundly impact bottom-up mass spectrometry analysis of proteins, since incomplete or non-specific proteolysis produces additional fragments that convolute data interpretation and obscure quantitative analysis. High hydrostatic pressure enhances enzyme activity via at least two mechanisms. First, high pressure decreases the activation volume of the reaction by compressing the solvent thus concentrating the enzyme, substrate, and its reaction product [1,2]. Secondly, high pressure drives the denaturation and unfolding of substrate proteins to expose previously inaccessible cleavage sites. While the positive effects of temperature and pressure on the activity of several proteases are known [3-5], the potential synergy of elevated temperature with pressure has not been fully characterized, particularly in terms of real-time kinetic studies within a context of proteomic sample preparation. Using a high pressure optical cell coupled to a programmable high pressure generator, the effects of elevated pressure (0-60 kpsi) and temperature (23°C to 55°C) on the rates at which trypsin, chymotrypsin, and endoproteinase Lys-C digest their respective synthetic chromogenic substrates, were investigated.