Improved proteolytic digestion under high pressure cycling: rapid digestion with improved sensitivity and sequence coverage.

摘要

Our data strongly suggest that use of pressure cycling technology (PCT) increases the recovery of observed peptides and improves sequence coverage, while also reducing sample preparation time. The effects of pressure cycling on trypsin proteolysis are, in part, substrate protein-specific, and can result in significant improvements in digestion and quantitative recovery of peptides from difficult-to-digest proteins that may be underrepresented in samples prepared using traditional workflows. Digestion of model proteins with trypsin for 90 minutes under pressure cycling conditions at 20,000psi shows significantly increased signal intensity and improved sequence coverage, compared to controls incubated for 90 minutes at ambient pressure. The improved digestion in PCT-treated samples is evident in samples prepared in 50mM ammonium bicarbonate with or without the addition of acid-labile detergent (0.05% RapiGest). Addition of 10% n-Propanol serves to further improve the PCT digest without compromising trypsin specificity. Pressure-accelerated digestion with trypsin alone was comparable to, or better than, sequential digestion with lys-C and trypsin, suggesting that under the conditions used here (PCT, urea-free buffer, model proteins), trypsin alone is sufficient for optimal digestion. However, it is likely that for samples in urea-containing lysis buffers, the benefit of including a lys-C step may be significant, as this enzyme is far more active in urea than is trypsin. In addition, we show that PCT can be used to accelerate the specific activity of other enzymes, including chymotrypsin, Lys-C, Glu-C, and Tryp-N without compromising enzyme specificity. Pressure Cycling has previously been shown not to cause an increase in non-specific chemical modifications of proteins such as methionine oxidation and asparagine or glutamine deamidation, which, combined with the specificity results presented here, strongly suggests that there are few, if any, drawbacks to incorporating PCT into current Proteomics workflows.