Vývoj enzymatických reaktorů a jejich využití ve strukturní proteomice.

Kalaninová, Zuzana

5 ABSTRACT (IN ENGLISH) Mass spectrometry (MS) has become indispensable in structural biology, enabling not only protein identification and quantification but also the investigation of higher-order structure and conformational dynamics. Among structural MS techniques, hydrogen/deuterium exchange MS (HDX-MS) holds a unique position due to its ability to probe protein flexibility and dynamics in solution. This doctoral thesis focused on the development and optimization of sample processing workflows in HDX-MS, particularly proteolytic strategies tailored for challenging targets such as highly stable antibodies, heavily modified proteins, membrane-associated proteins, and sequences resistant to conventional proteases. Special attention was given to the prolyl endopeptidase AnPEP, evaluated for online digestion. During this work, a previously unrecognized preference for reduced cysteine residues was discovered, a feature later identified as a common trend in a whole class of post- proline cleaving enzymes. The potential of immobilized AnPEP, either alone or in combination with other acidic proteases, was then demonstrated on the proline-rich prion protein. In parallel, additional immobilized protease columns, comprising single or mixed acidic proteases, were tested for the digestion of therapeutic antibodies....

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