Pressure-Enhanced Enzymes Application Notes

Pressure-Enhanced Enzymes Application Notes

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Pressure-Accelerated Protein Digestion with Endoproteinase Lys-C

Introduction
Pressure Cycling Technology (PCT) has been proven to accelerate enzymatic protein digestion. The positive effect of PCT on trypsin digestion is well established [1-4] for improved sequence coverage, higher recovery and significantly reduced digestion times. 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, chymotrypsin and lysozyme has been reported [6-10].

Proteolysis-PrEP: Optimization of Pressure Cycling Conditions for Protein Digestion with Trypsin in Solution

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].

Proteolysis-PrEP (Lysozyme): Effect of Pressure Cycling on Lysozyme Activity

Introduction
Pressure cycling technology (PCT) has been proven to accelerate enzymatic protein digestion. For example, the effect of PCT on trypsin digestion has been demonstrated by several laboratories. They report that digestion times can be reduced from hours to minutes [1, 2]. Not only has PCT been shown to accelerate and improve protein digestion in solution, but it also can accelerate the digestion by trypsin of proteins in polyacrylamide gel slices [3]. Additionally, the enhancing effect of PCT on the activity of several other enzymes, including Proteinase K, PNGase F, and Lys-C, has been reported [4, 5, 6].

Proteolysis-PrEP: Pressure-accelerated Activity of Endoproteinase Glu-C

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].

Proteolysis-PrEP: Activity and Specificity of Chymotrypsin Under Pressure Cycling Condition

Introduction
Pressure Cycling Technology (PCT) has been proven to accelerate enzymatic protein digestion. The positive effect of PCT on trypsin digestion is well established for improved sequence coverage, higher recovery and significantly reduced digestion times [1-5]. Additionally, the enhancing effects of PCT on the activity of other enzymes, including Proteinase K, PNGase F, Glu-C, Lys-C, and lysozyme have been reported [6-12].

Proteolysis-PrEP: Optimization of Pressure Cycling Conditions for Efficient Digestion of Native IgG

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.

Guidelines for Protocol Development:

Introduction
Pressure-enhanced proteolytic digestion exploits the ability of high hydrostatic pressure to promote protein denaturation and the access of proteolytic enzymes to their target sites. Pressure denaturation is fundamentally different from thermal denaturation as it occurs by virtue of hydration of hydrophobic residues and by water saturation of protein substrate cavities normally inaccessible to solvent. Pressure denaturation is more efficient for hydrophobic proteins, while some hydrophilic soluble proteins are reported to retain relatively compact conformation, even when saturated by water molecules [1-6].

Proteolysis-PrEP: Pressure-Accelerated Activity of Elastase Enzyme

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].

Proteolysis-PrEP: Pressure-Accelerated Protein Digestion by Thermolysin Enzyme

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 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].

Pressure-Accelerated Native Protein Deglycosylation by PNGase F Enzyme

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 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.

High Pressure-Accelerated Lys-C Digestion in the HUB880 Explorer: Rapid Digestion of Unreduced IgG Part I

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.

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.

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.

Pressure Cycling-Accelerated Digestion with Trypsin: Protein Modifications and Enzyme Specificity

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.

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.