PCT-HD (PCT-SWATH) Application Notes

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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.