Pressure-Enhanced Enzymes Application Notes

Pressure-Enhanced Enzymes Application Notes

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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, chymotrypsin, Glu-C, thermolysin, Proteinase K, and lysozyme, has been reported [5-11]. Pressure-accelerated deglycosylation by PNGase F of denatured glycoproteins has previously been shown [12]. 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 [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 enzyme activity, 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 deglycosylation of native proteins by PNGase F 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 deglycosylation with PNGase F.

Introduction
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 [6], chymotrypsin [7, 8], Glu-C [9], thermolysin [10], Proteinase K [11], PNGase F [12], and lysozyme [13], 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 [14]. 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.

Introduction

The benefit of high pressure incubation for enhanced Lys-C digestion of unreduced IgG, and the added benefit of reagents such as urea and sodium deoxycholate, is described in separate Application Notes [4, 19]. In the current Application Note we explore the effect of several organic solvents on pressure-enhanced digestion, in order to provide the best set of starting conditions for high pressure-enhanced Lys-C digestion of disulfide-intact IgG in the presence of these reagents. These conditions are likely to be similar for digestion of other hard-to-digest proteins, such as those containing hydrophobic transmembrane domains. The current application focuses on digestion at constant high pressure (not pressure cycling) in the HUB880 Explorer.

Introduction

The benefit of high pressure incubation for enhanced Lys-C digestion of unreduced IgG, and the added benefit of reagents such as urea and sodium deoxycholate, is described in separate Application Notes [4, 19]. In the current Application Note we explore the effect of several organic solvents on pressure-enhanced digestion, in order to provide the best set of starting conditions for high pressure-enhanced Lys-C digestion of disulfide-intact IgG in the presence of these reagents. These conditions are likely to be similar for digestion of other hard-to-digest proteins, such as those containing hydrophobic transmembrane domains. The current application focuses on digestion at constant high pressure (not pressure cycling) in the HUB880 Explorer.

Introduction

The goal of the current Application Note is to provide the best set of starting conditions for high pressure-enhanced Lys-C digestion of disulfide-intact IgG. These conditions are likely to be similar for digestion of other hard-to-digest proteins, such as those containing hydrophobic transmembrane domains. The current application focuses on digestion at constant high pressure (not pressure cycling) in the HUB880 Explorer.

Introduction

The benefit of high pressure incubation for enhanced Lys-C digestion of unreduced IgG, and the added benefit of reagents such as acetonitrile or N-propanol is described in separate Application Notes [4, 5]. In the current Application note we explore the effect of urea and sodium deoxycholate on pressure-enhanced digestion, in order to provide the best set of starting conditions for high pressure-enhanced Lys-C digestion of disulfide-intact IgG in the presence of these reagents. These conditions are likely to be similar for digestion of other hard-to-digest proteins, such as those containing hydrophobic transmembrane domains. The current application focuses on digestion at constant high pressure (not pressure cycling) in the HUB880 Explorer.