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Atmospheric pressure chemical ionization mass spectrometry at low flows: construction and applications
Strmeň, Timotej ; Cvačka, Josef (advisor) ; Sýkora, David (referee) ; Polášek, Miroslav (referee)
(EN) The first part of the thesis describes the ionization reactions in the APCI source in the presence (enclosed configuration) and absence (open configuration) of the ion source housing. Commercial APCI source was modified and used in enclosed, as well as open configuration. All other parameters were kept the same (including the source geometry). In the positive mode, the biggest differences were observed for non-polar analytes dissolved in aprotic solvents like toluene, chloroform and carbon disulfide. Protonated species were dominant in the open configuration, while radical cations were mostly present in the enclosed configuration. The excessive protonation in open ion source was caused by the diffusion of water vapor molecules into the ionization region from the atmosphere. Water vapor molecules were also responsible for the formation of an ion [M + 19]+ from alkynes in the open configuration. The fragmentation study confirmed that the ion [M + 19]+ was a 2-methylketone formed by the addition reaction. The formation of such artifacts can pose problems in qualitative analysis. On the other hand, the ion [M + 55]+ was observed in the enclosed ion source as a reaction product of unsaturated compounds with acetonitrile solvent molecules. Its fragmentation can be used for the double bond...
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Recombinant preparation of DNA binding domain of transcription factor TEAD4
Zákopčaník, Marek ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...
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Structural characterization of dsDNA and transcription factor interactions
Kadavá, Tereza ; Novák, Petr (advisor) ; Hubálek, Martin (referee)
Transcription factors from TEAD protein family play an important role in various eukaryotic regulatory processes. They are partly responsible for cell differentiation and apoptosis due to their involvement in the Hippo signalling pathway. Scientific interest in this protein family is mainly because they are associated with carcinogenesis. DNA-binding domain of TEAD transcription factors interacts with M-CAT element of double-stranded DNA, which is present also in enhancer and exon of C-MYC gene. Interaction between TEAD1 DNA-binding domain and C-MYC dsDNA was investigated in this bachelor's thesis mainly by ion mobility in connection with mass spectrometry using native nESI ionization. For the further analysis of TEAD1-DBD and its complexes with dsDNA, the protein was firstly recombinantly expressed by the optimized protocol in our laboratory. Investigation of TEAD1-DBD and its complexes was done using two IM-MS activations. Firstly, the collision-induced unfolding was performed and afterwards perturbation of the sample by preheating nESI emitter was done. The first activation led to the collision- induced unfolding of some charge states of TEAD1-DBD protein and both protein-DNA complexes. The second activation performed by heating of nESI emitters led to temperature melting of both dsDNA and...
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Study of derivatization reactions for ESI-MS analysis of hardly ionizable rhenium aryl chlorocomplexes
Vlk, Mikuláš ; Štícha, Martin (advisor) ; Kozlík, Petr (referee)
Mass spectrometry with electrospray ionization is an excellent method for structural analysis of coordination compounds with outstanding sensitivity and selectivity. However, it fails to detect some low-polar rhenium complexes. This master thesis describes derivatization method of non-ionizable rhenium complexes with 1,2-dihydroxybenzene and 2,3- dihydroxytoluenene. Fragmentation mechanisms and structure of prepared complexes was studied using high resolution mass spectrometry and collision-induced dissociation (CID). Furthermore, density functional theory (DFT) computational method was used for prediction of bond cleavage based on bond lengthening.
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Quantification of procalcitonin in human serum by affinity surfaces and mass spectrometry
Dvořák, Josef ; Pompach, Petr (advisor) ; Kozlík, Petr (referee)
Sepsis is a worldwide health condition. It is caused by disproportionatly large immunity system response for patogene presence. Fast and accurate diagnosis of sepsis can make difference between life and death. For clinical diagnosis of sepsis is used serum protein procalcitonin (PCT) as biomarker. Procalcitonin concentration level in patient bloodstream increases up to thousand times in short time period. Severity of sepsis can be determined by correlation with its concentration in bloodstream. To determine PCT level in patient bloodstream a broad variety of specialized instrument is used. All methods have same principle of measurement - PCT-antibody interaction. Procalcitonin level is then quantified by calibration curve method. During these measurements non-specific protein-antibody interactions can occur and distort the quantification of PCT level. Mass spectrometry due to its properties comes into consideration as an alternative method, that can be used for PCT determination. Aim of this diploma thesis was in situ enrichment of PCT on surface modified affinity carriers with immobilized antibody against PCT. These carriers are compatible with matrix assisted laser desorption/ionization time of flight mass spectrometry. Mass spectrometry provides mass spectrum, where PCT and other signals...
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The novel combinations of experimental approaches: mass spectrometry (MS) and photo-induced surface labelling, electron release (PIER), or cross-linking (PIXL)
Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
Countless electron transport/transfer (ET) processes occur in living organisms every day. Therefore, their study is a crucial field of modern structural and functional proteomics. In many cases model proteins like azurin from P. aeruginosa are utilised in experiments. This blue copper protein is favoured due to a characteristic absorbance maximum at 630 nm in Cu(II) redox state of the central Cu atom. During its oxidation to Cu(I) state the A630 value decreases allowing UV-Vis detection of ET reaction progress. We have introduced a structural photoinducible analogue of canonical amino acid Met - L-2-amino-5,5-azihexanoic acid (photo-Met) - into azurin structure to study oligomerization in solution via photo-induced cross-linking (PIXL). Using previously optimised protocols for recombinant expression in E. coli B834 we have inserted photo-Met into azurin moieties: wild type azurin and Az2W mutant where two adjacent W residues with confirmed role in electron hopping across protein-protein interface are present. The incorporation percentage of photo-Met in analysed samples was determined after SDS-PAGE and in-gel protease digestion via MALDI-TOF MS. PIXL was employed to study azurin-azurin interaction and oligomerization under different total concentrations of protein (in range of 15-300 µM). The...
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The use of protein radical footprinting by Togni reagents in structural biology
Fojtík, Lukáš ; Kukačka, Zdeněk (advisor) ; Kolenko, Petr (referee)
Structural proteomic methods such as an ion mobility mass spectrometry, chemical cross- linking or covalent labeling have been established as powerful tools for structural studies of biomolecules in general. These methods have significantly contributed to the expansion of our knowledge about biomolecular functions, their dynamics and molecular interactions and therefore led to the understanding of important biological processes occurring in a cell. We decided to spread these methods and we developed a new radical labeling technique relaying on Fluor-alkyl radicals that does not require a laser dissociation pf hydrogen peroxide. We exploited the potential of Togni reagents to form Fluor-alkyl radicals by reducing agent under native conditions. The induction of Fluor-alkyl radicals was triggered by ascorbic acid and the labeling pulse was stopped by tryptophan. The modified proteins were analyzed by top down or bottom up approach using high resolution mass spectrometry. In case of top down approach, several fragmentation techniques (CID-ECD, ETD) were tested for protein analysis while in case of bottom up approach the analyzed proteins were digested by trypsin and separated on reverse phase column online coupled to mass spectrometer. As the model biomolecules we chose a 20 proteinogenic amino acids...
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Study of microplastic sorption properties
Hlaváčková, Hana ; Kučerík, Jiří (referee) ; Řezáčová, Veronika (advisor)
There is currently an effort to replace conventional plastics with biodegradable products that will have the ability to replace these substances. One of the possible biodegradable felt, which is currently being studied in detail, is also poly (3-hydroxybutyrate), which is produced by bacteria. This bachelor thesis deals with the sorption of selected substances on poly(3-hydroxybutyrate) microparticles. Substances whose contamination of water sources is a significant problem today have been selected for this work. Specifically, the sorption of the nonsteroidal anti-inflammatory substance ibuprofen, the triazole fungicide tebuconazole and the most common drug caffeine on PHB particles, which fall in the size of the microplastic group, were studied. The method of liquid chromatography with mass detection was used for analyzes.
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