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Sample preparation for analyzing oligomeric forms of amyloid beta by mass spectrometry
Mrkvičková, Simona ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
Alzheimer's disease is the most widespread chronic and progressive neurodegenerative disease. The main problem of the disease is the reduction and damage of nerve cells. This leads to loss of brain mass and memory impairment. The disease mainly affects the elderly, for whom it is the most common cause of dementia. Around 50 million people suffer from this disease worldwide, and the number is growing. Alzheimer's disease thus represents a serious public health problem. The pathogenesis of this disease is controlled by the production and deposition of amyloid beta peptide. This peptide tends to aggregate. Its lower oligomeric forms are water-soluble and have a toxic effect on nerve cells. Amyloid beta oligomers therefore represent a possible target for the diagnosis of Alzheimer's disease. Oligomeric forms of beta amyloids have a non-covalent character, which can negatively affect their stability during analyzes in model and biological samples. In this bachelor thesis, suitable conditions for the preparation of model amyloid beta samples were sought. It was observed how the distribution of oligomeric forms changes under the influence of solvents, salts and other additives. The effect of pH on the incubation of amyloid beta in ammonium acetate and water was also investigated. Finally, a method for the...
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Use of separation and mass spectrometric techniques in the diagnosis of Alzheimer's disease
Lebertová, Lucie ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee)
This thesis focuses on the preparation of covalently cross-linked oligomeric forms of amyloid beta and their analysis by SDS-PAGE and MALDI-TOF. The covalently cross-linked oligomeric forms of Aβ(1-42) were formed by chemical and enzymatic reactions. The chains of amyloid beta are crossed over the amino acid residue of tyrosine to form a covalent bond. Such covalent cross-linking stabilizes the oligomeric structure, which allows for improved analysis of amyloid beta oligomers. The development of methods for the detection and quantification of amyloid beta oligomers may be of considerable benefit for the diagnosis of Alzheimer's disease, as it is the oligomeric forms of amyloid beta that are most strongly associated with Alzheimer's disease pathology. In the theoretical part, Alzheimer's disease is described with the above-mentioned problems in early diagnosis and therapy. In addition, background information on amyloid beta is given, and analytical methods for the separation and detection of amyloid beta used in research are described. The experimental part deals with the optimization of analytical methods MALDI-TOF and SDS-PAGE. In the MALDI-TOF, 2,5-dihydroxybenzoic acid and sinapic acid matrices were selected as suitable matrices for the determination of Aβ(1-42). The most suitable method of...
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A new method for double bond characterization in lipids by ultraviolet photodissociation mass spectrometry
Rumlová, Barbora ; Strmeň, T. ; Cvačka, Josef
Lipids are structurally diverse biomolecules with vital functions in biological systems. Some of these functions are closely associated with the specific location of the carbon-carbon double bond in the lipid acyl chains. The chain length and the number of unsaturated carbon-carbon bonds can be determined using conventional MS/MS-based structural elucidation methods employing low or higher energy collision-induced dissociation. However, these dissociation techniques do not provide more subtle structural details, for example, the position of unsaturated bonds. Another type of activation method – ultraviolet photodissociation can be used for this purpose. In this study, a new method for characterization of double bond location in lipids acyl chain was developed using 193 nm ultraviolet photodissociation implemented on Orbitrap Fusion Lumos mass spectrometer. This approach is based on the derivatization of the double bond with bis-(5-iodo-[2]pyridyl)-disulfide, subsequent specific cleavages provide a unique diagnostic pair bearing information about the double bond position.
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