Introduction
The positive effect of Pressure Cycling Technology (PCT) on digestion with trypsin is well established [1-4], 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, Proteinase K, PNGase F, chymotrypsin, Glu-C and lysozyme has been reported [5-11].
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 [12]. 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 digestion, as long as the level of applied pressure is below the level at which the enzyme itself is denatured and inactivated. In addition, under certain conditions, hydrostatic pressure can have a positive effect on enzyme activity itself, independent of substrate conformation.
Here we report that protein digestion by thermolysin enzyme is accelerated when the reaction is carried out under pressure cycling conditions. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced thermolysin digestion.

Introduction
The positive effect of Pressure Cycling Technology (PCT) on trypsin digestion is well established [1-6], and has been shown to result in improved sequence coverage, higher recovery and significantly reduced digestion times. Additionally, the enhancing effect of PCT on the activity of several other enzymes, including Lys-C, Proteinase K, PNGase F, chymotrypsin and lysozyme has been reported [7-13].

Introduction
The positive effect of Pressure Cycling Technology (PCT) on trypsin digestion is well established [1-4], and has been shown to result in improved sequence coverage, higher recovery and significantly reduced digestion times. Additionally, the enhancing effect of PCT on the activity of several other enzymes, including Lys-C, Proteinase K, PNGase F, chymotrypsin, Glu-C, thermolysin, and lysozyme has been reported [5-12].
Pressure, heat, and many different 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 [13]. 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 digestion, as long as the level of pressure that is applied is below the level at which the enzyme itself is denatured and inactivated. In addition, under certain conditions, hydrostatic pressure can have a positive effect on enzyme activity, independent of substrate conformation.
Here we report that protein digestion by elastase enzyme is accelerated when the enzyme reaction is carried out under pressure cycling conditions. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced Elastase digestion.

Introduction
Pressure Cycling Technology (PCT) has been proven to accelerate enzymatic protein digestion. The positive effect of PCT on trypsin digestion has been demonstrated by several laboratories [1, 2, 3] who have reported improved sequence coverage, higher recovery and significantly reduced digestion times when the trypsin reactions were carried out under pressure. Not only has PCT been shown to accelerate and improve digestion in solution, but it can also accelerate in-gel trypsin digestion [4, 5]. Additionally, the enhancing effect of PCT on the activity of several other enzymes, including Proteinase K, PNGase F, Lys-C and lysozyme has been reported [6, 7, 8, 9].

Introduction

PCT has been proven to accelerate enzymatic protein digestion and the positive effect of PCT on digestion by both trypsin and Lys-C is well established [1-4]. Additionally, the enhancing effects of PCT on the activity of enzymes including Proteinase K, PNGase F, chymotrypsin, and lysozyme have been reported [5-8]. High hydrostatic pressure accelerates protein digestion via a combination of two mechanisms. The first is the pressure-induced partial denaturation/unfolding and hydration of the target proteins, which leads to better access of the enzyme to its target sites. The second mechanism is less well understood, but relies on the increase in enzyme activity observed for some enzymes at elevated pressure. This second mechanism is likely due, at least in part, to the positive effect of hydrostatic pressure on all reactions that involve hydrolysis. The combination of the two pressure-based mechanisms, one acting on the enzyme and the other acting on the substrates, leads to significantly accelerated digestion. Pressure induces protein denaturation, but the pressure-perturbed proteins assume conformational forms that are different from those caused by thermal or chemical treatment [9]. Pressure-induced denaturation, when combined with chaotropes and/or detergents that are often used in standard (atmospheric pressure) digestion reactions, can potentially lead to denaturation and inactivation of the enzyme itself. Thus, it is important to determine which reagents, and at what concentrations, are compatible with pressure cycling-enhanced digestion protocols. Such reagents are often necessary when working with native proteins where disulfide links have not been reduced and complex tertiary structure of the substrate protein may severely limit enzyme access to target sites. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced digestion of hard-to-digest proteins, such as unreduced (disulfide-intact) IgG.