Improving digestion efficiency under H/D exchange conditions with activated pepsinogen coupled columns

The ability to analyze localized amide hydrogen/deuterium (H/D) exchange kinetics of a protein is highly dependent on the digestion efficiency of the enzyme being used to generate a collection of labeled peptides. Pepsin is one of the most commonly used acid proteases in amide H/D exchange experiments due to its broad specificity and robustness at “slow exchange” conditions of low temperature and pH. The chemistry used to immobilize pepsin to POROS AL-20 beads is optimal under neutral pH conditions but acid proteases such as pepsin are irreversibly denatured above pH 5 so coupling must be performed under suboptimal conditions. Thus, in the current study we report a technique to improve the digestion efficiency for amide H/D exchange by immobilizing pepsinogen to POROS AL-20 support under optimal coupling conditions of pH 6.7 where pepsinogen is stable and subsequently converting the coupled pepsinogen into active pepsin. This activated pepsinogen column demonstrated a higher specific activity at both 25 °C and 0 °C than an identically coupled pepsin column and gave better peptide coverage for cytochrome C and manganese superoxide dismutase (MnSOD) improving our amide H/D exchange data for these proteins. These results were reproducible for three independently coupled and activated pepsinogen columns. Protein assays demonstrated that more enzyme was bound to the POROS AL-20 resin coupled with pepsinogen at pH 6.7 than pepsin at pH 5.