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Mass spectrometry in proteomics: structural biology and clinical applications
Pavlásková, Kateřina ; Šulc, Miroslav (advisor) ; Obšilová, Veronika (referee) ; Hernychová, Lenka (referee)
Mass spectrometry (MS) is a rapid, specific and very sensitive analytical method with a broad spectrum of proteomic applications such as protein identification and sequencing, 3D protein structure characterization or study of protein-protein interaction. The introduction of two ionization techniques in late 1980's that are able to ionize the large biomolecules such as proteins, oligosaccharides or nucleic acids with no or low fragmentation has started the rapidly expanding field of MS-based proteomics. The presented thesis was aimed at the application of mass spectrometric approaches to answer several proteomic questions. Firstly we have employed the chemical cross-linking in combination with MS analysis to solve the 3D structure and protein-protein interactions of three model systems: (1) homodimeric human regulatory protein 14-3-3, (2) model of 14-3-3 and regulatory domain of tyrosine hydroxylase, and (3) system of two membrane proteins, cytochrome P450 2B4 and cytochrome b5, involved in xenobiotics biotransformation. This approach works in aqueous solutions under physiological conditions and thus preserves native structure of the investigated proteins. The second part of the thesis was focused on MS identification of proteins/peptides in fungal spores of Aspergillus and Pseudallescheria...
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Study of modified amino acid incorporation into cytochrome b5
Koberová, Monika ; Hodek, Petr (advisor) ; Pavlásková, Kateřina (referee)
A cytochrome b5 (cyt b5) can influence cytochrome P450 (CYP)-dependent reactions. In consequence of these reactions cytochrome b5 can participate in substance activation (for example drugs, carcinogens) or it can influence proportions of formed metabolites. A mechanism of cyt b5 action has not been fully explained yet. Elucidation of protein-protein interactions in monooxygenase system could explain of the mechanism of cyt b5 action. To study these interactions by using cross-linking techniques is necessary to prepare photolabile cyt b5, which after photoactivation generated higly reactive intermediates which can create a complex with nearby components of the monoogynesase system. This thesis describes how was developed the method for the production of recombinant cyt b5 with modified amino acids. Cyt b5 was expressed in a bacterial strain E. coli BL-21 (DE3) Gold. Before the expression induction, cells were transformed into the limiting medium (DMEM-LM) which did not contain L-leucine and L-methionine. The limiting medium was supplemented by deuterated amino acid d3-methyl-L-methionine and D,L-Leucine. Expressed cyt b5 was isolated and incorporation of d3-methyl-L-methionene has been verified by mass spectrometry. Cyt b5 was obtained mainly as the apoprotein (apo-cyt b5). That is why in this...
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