|
|
Preparation of proteases for protein structure studies.
Jirečková, Barbora ; Man, Petr (advisor) ; Vaňková, Pavla (referee)
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...
|
|
|
Development of alternative proteolysis for protein identification
Jirečková, Barbora ; Man, Petr (advisor) ; Hnízda, Aleš (referee)
5 Abstract Methods of protein research based on mass spectrometry have become increasingly significant in the last few years. The proteolytic step is essential in a bottom-up approach, determining the success and relevance of identification and characterization of a protein and also the sequence coverage and spatial resolution of HDX-MS protocols. Conventionally used proteases however have their limitations and therefore efforts are being made to find alternatives that have, above all, different cleaving preferences and activity profiles. AnPEP protease (Aspergillus niger prolyl endoprotease) has the advantages of both characteristic cleavage in the presence of proline (unlike many other proteases) and easy accessibility due to its commercial obtainability in large quantities. This thesis examines the optimization of cleavage using AnPEP protease obtained from commercially prepared Gluten Rid with Tolerase G. Firstly AnPEP cleavage was tested on model protein (bovine carbonic anhydrase 2) under various conditions (temperature, pH, concentration of protease on a carrier or in solution). The objective was to find out how various conditions influence cleavage efficiency and preferences of this protease. It was discovered that AnPEP protease is suitable for hydrogen/deuterium exchange coupled to mass...
|
guest ::
