High Pressure Optical Spectroscopy

High Pressure Optical Spectroscopy

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INTRODUCTION

The rate, specificity, and efficiency of proteolytic digestion profoundly impact bottom-up mass spectrometry analysis of proteins, since incomplete or non-specific proteolysis produces additional fragments that convolute data interpretation and obscure quantitative analysis. High hydrostatic pressure enhances enzyme activity via at least two mechanisms.  First, high pressure decreases the activation volume of the reaction by compressing the solvent (e.g. water is compressed 11.5% at 60 kpsi) thus concentrating the enzyme, substrate, and its reaction product [1,2].  Secondly, high pressure drives the denaturation and unfolding of proteins to expose previously inaccessible cleavage sites.  While the positive effects of temperature and pressure on the activity of several proteases are known [2-5], the potential synergy of elevated temperature with pressure has not been fully characterized, particularly in terms of real-time kinetic studies within a context of proteomic sample preparation. Using a high pressure optical cell coupled to a programmable high-pressure generator, the effects of elevated pressure (0-60 kpsi) and temperature (23°C to 55°C) on the rate at which lysyl endoproteinase (Lys-C) digests a synthetic chromogenic substrate, was investigated.

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.