Proteomics Under Pressure: Rapid Extraction and Digestion in a Single Tube.

摘要

Pressure cycling has been shown to significantly improve proteolysis with a number of enzymes. High hydrostatic pressure acts synergistically with chaotropes and organic solvents to boost the effects of these chemicals on protein denaturation and digestion. These effects not only improve digestion efficiency and save significant time, but also allow the use of much lower concentrations of denaturants, which can simplify and improve downstream applications. While such effects are clearly beneficial, care must be taken in development of PCT-enhanced digestion methods to avoid impairment of enzymatic activity due to denaturation of the enzyme itself. The following points illustrate some of our recent findings: 1. Chymotrypsin activity can be significantly enhanced by performing digestion using cycled pressure. 2. Under cycled pressure, 0.5 M Guanidine-HCI inhibits trypsin but not chymotrypsin activities. 3. Under cycled pressure, 4percent HFIP enhances trypsin but inhibits chymotrypsin activities. 4. Pressure levels above 20,000 psi exhibit negative effect on trypsin activity. 5. Under cycled pressure, trifluoroethanol (TFE) below 15percent enhances trypsin but inhibits chymotrypsin activities (data not shown). 6. Under cycled pressure, both methanol and urea can be included in trypsin digests at low concentration, but become rapidly inhibitory at higher concentrations. 7. Pressure cycling can be used to enhance cell lysis and accelerate protein digestion in the same sample container while minimizing sample handling and potential loss of analytes.