Preparation of proteases for protein structure studies.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is a method allowing the study of protein structure and dynamics. Its spatial resolution is given by the proteolysis step that is included in the HDX-MS workflow. Most widely used pepsin has however some limitations and use of a single protease often does not provide optimal spatial resolution. Several publications have emphasized the importance of the alternative proteases nepenthesin-2 (Nep-2) and aspergillopepsin (XIII) cleaving, in contrast to pepsin, after basic amino acids. In studies targeting proline-rich proteins, another enzyme, prolyl endoprotease from Aspergillus niger (AnPEP), is gaining importance. This work focuses on the characterization of immobilized AnPEP in combination with pepsin, aspergillopepsin or Nep-2 for their application in HDX-MS. First, columns with only one protease were tested on a set of model proteins. It was found that immobilized AnPEP did not have optimal cleavage characteristics compared to the other proteases. In order to combine the advantages of the proteases mentioned above, the model proteins were digested using columns with AnPEP coimmobilized with pepsin, Nep-2 or XIII and also using two protease columns in series (always AnPEP column with pepsin, Nep-2 or XIII column in both..