Pressure-Enhanced Enzymes Application Notes

Pressure-Enhanced Enzymes Application Notes

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Pressure Cycling-Accelerated Digestion with Trypsin: Effect of Urea on Digestion at Pressure

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 Cycling-Accelerated Digestion with Trypsin: Digestion of Native Proteins in Whole Tissue Lysate

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].

III. Effect of High Pressure and Temperature on Papainase Digestion Rate

INTRODUCTION
High hydrostatic pressure (HPP) has been used for decades in the food industry for the inactivation of microbes and of the oxidases responsible for the browning of fruits and vegetables [5].  In biotechnology, HHP has been shown to accelerate the activity of proteases used for the digestion of proteins prior to mass spectrometry.  While the positive effects of temperature and pressure on the activity of several proteases are known [6,7], the potential synergy of elevated temperature with pressure has not been fully characterized, particularly in terms of real-time kinetic studies.  Using a high pressure optical cell coupled to a programmable high pressure generator, the effects of elevated pressure (0-60,000 psi) and temperature (22°C to 60°C) on the rate at which papainase digests a synthetic substrate was investigated. 

High Pressure-Accelerated Lys-C Digestion in the HUB880 Explorer: Rapid Digestion of Unreduced IgG Part III. Effects of Urea and Sodium Deoxycholate

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.

High Pressure-Accelerated Lys-C Digestion in the HUB880 Explorer: Rapid Digestion of Unreduced IgG Part II. Effect of Acetonitrile, N-Propanol, and Methanol

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.