Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium (H/D) exchange coupled to mass spectrometry is nowadays routinely used to probe protein interactions or conformational changes. The method has many advantages, e.g. very low sample consumption, but offers limited spatial resolution. One way to higher resolution leads through the use of different proteases or their combinations. In the present work we describe recombinant production, purification and use of aspartic protease zymogen from Rhizopus chimensis, protease type XVIII (EC 3.4.23.6), commonly referred to as rhizopuspepsinogen (Rpg). The enzyme was expressed in Escherichia coli, refolded and purified to homogeneity. A typical yield was approximately 100 mg of pure enzyme per 1 L of original bacterial culture. The kinetics of protease activation, i.e. removal of the propeptide achieved by autolysis in an acidic environment, was followed by mass spectrometry. The digestion efficiency was tested for the protease in solution as well as for the immobilized enzyme. Apomyoglobin was successfully digested under all conditions tested and the protease displayed very low or no autodigestion. The results outperformed those obtained with commercial protease where the digestion of apomyoglobin was incomplete and accompanied by many contaminating peptides. Taken together, the recombinant protease type XVIII can be considered as a new and highly efficient tool for H/D exchange followed by mass spectrometry. Copyright 2009 John Wiley & Sons, Ltd.