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].

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.