Pressure-Enhanced Enzymes Application Notes

Pressure-Enhanced Enzymes Application Notes

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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].

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].

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].

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
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.