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].

The PCT-HD system is a combination of two of PBI’s innovative sample preparation tools; the NEP2320-Enhanced Barocycler and PCT MicroPestles. This unit can generate a maximum pressure of 45 kpsi (the original NEP2320 Barocycler is rated to 35 kpsi) and can be heated to temperatures up to 95⁰ C. The higher pressure and temperature range give the NEP2320-Enhanced broad applicability in proteomics, enzymology and other fields of research.

High hydrostatic pressure accelerates protein digestion via a combination of two mechanisms. The first is the pressure-induced partial denaturation/unfolding and hydration of the target proteins, which leads to better access of the enzyme to its target sites. The second mechanism is less well understood, but relies on the increase in enzyme activity observed for some enzymes at elevated pressure, possibly resulting from the effect of pressure on hydrolysis. The combination of the two pressure-based mechanisms, one acting on the enzyme and the other acting on the substrates, leads to significantly accelerated digestion, but may need to be optimized for each enzyme and reagent system, to prevent pressure-induced denaturation and loss of activity of the enzyme itself. To this end, the effect of sodium deoxycholate (DOX) on PCT-accelerated trypsin and lys-C digestions, has been evaluated.

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].

The PCT-HD workflow (also known as PCT-SWATH^Tm or PCT-ABLE) is a combination of two of PBI’s innovative sample preparation tools; the 2320EXT Barocycler and PCT MicroPestles [1-4]. Together they utilize pressure cycling technology (PCT) to extract and digest proteins from biopsy-size tissue samples. PCT has been proven to accelerate enzymatic protein digestion; the positive effect of PCT on digestion by both trypsin and Lys-C has previously been demonstrated [2-9]. High hydrostatic pressure improves protein digestion via a combination of two mechanisms; one acting on the enzyme and the other acting on the substrates, leading to significantly accelerated and highly reproducible digestion (Figure 1). The method can be adjusted for digestion with trypsin alone, lys-C alone, or a combination of both enzymes. Additional optimization may be required when using non-conventional proteolytic enzymes. PCT-HD can be carried out using conventional enzymes or boutique enzymes designed for higher temperature methods [4].