INTRODUCTION

Papainase is a cysteine protease derived from Carica papaya and is commonly used in the food industry as a meat tenderizer.  In biotechnology, the enzyme is frequently used for the limited proteolysis of IgG to generate specific Fab and Fc fragments.  Medicinally, papainase has been used to liquify the contents of herniated vertebral discs so that their contents can be removed by aspiration [1], in wound debridement [2], and for the removal of dental caries [3] and exhibits antihelmintic properties [4].

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. 

Introduction

Here we report that 90 minute pressure-accelerated trypsin digestion of whole tissue lysate is comparable to, or better than, standard overnight digestion. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced trypsin digestion of complex samples.

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.

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.