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Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

Diplomová práce pojednává o zavedení a optimalizaci metody ELISA pro průkaz a kvantifikaci specifických anti-CAI protilátek v lidském séru. Test by měl sloužit pro vyšetření pacientů s hematoonkologickým onemocněním. Byla provedena optimalizace podmínek stanovení a tyto podmínky byly poté použity pro proměření pěti neznámých vzorků séra od pacientů s mnohočetným myelomem, kteří byli léčeni vysokodávkovým ozařováním s následnou transplantací kmenových buněk. Teoretická část shrnuje poznatky o jednotlivých druzích EIA metod, o indikátorech používaných pro imunoanalytické metody, o možnostech, jak zvýšit citlivost detekce. V závěrečné kapitole jsou zmíněny přístroje pro automatizaci imunoenzymatických metod., The thesis discusses the implementation and optimization of ELISA methods for the detection and quantification of specific anti-CAI antibodies in human serum. The test should be used for the evaluation of patients with hematooncological disease. The optimization of assay conditions was performed and these conditions were then used for the measurement of five unknown serum samples of multiple myeloma patients treated by high dose irradiation with subsequent stem cell transplantation. The theoretical part summarizes information on various types of EIA methods, the indicators used for immunoassays, ways how to increase the detection sensitivity. The final chapter describes devices for automation of immunoenzymatic methods, Fakulta chemicko-technologická