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Development of chomatography-mass spectrometry technique for separation and identification of glycopeptides applicable in clinical practice
Petroušek, Filip ; Ječmen, Tomáš (advisor) ; Kukačka, Zdeněk (referee)
Glycoproteins are commonly found in organisms and play an important role in both physiological and pathological conditions. In glycoproteins, different glycans may be present at the same glycosylation site and altered glycosylation of proteins may be a manifestation of disease. As a result, they can be used as markers in diagnostics. For the analysis of glycoproteins, their cleavage into glycopeptides, which are then identified, is often used. Liquid chromatography coupled to mass spectrometry is a widely used method for the study of glycopeptides and hydrophilic interaction chromatography (HILIC) can be used for the separation of glycopeptides. This type of chromatography allows separation of glycopeptides based on the properties of both the peptide and the bound glycan. The first aim of this work was to determine the extent to which the charge of the peptide backbone of glycopeptides affects their separation by HILIC. Penta-HILIC and HILIC-B columns were used for this purpose. Penta-HILIC contains a polyhydroxyl stationary phase, while the stationary phase of HILIC-B consists of silica gel modified with positively charged aminopropyl groups. IgG1 and IgG2 glycopeptides were separated on both columns. To determine the extent of the effect of charge and hydrophobicity during separation on HILIC-B,...
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Generation of synthetic modified mRNAs encoding cell cycle regulators
Petroušek, Filip ; Koblas, Tomáš (advisor) ; Vašková, Michaela (referee)
One of the modern therapeutic approaches is the application of in vitro transcribed mRNA into the target cells to produce the required target proteins. This method has prooved to be more effective than therapeutic application of proteins prepared in advance, because their insertion into the organism lead to denaturation. The main task of this work was to prepare mRNA in vitro and mRNA transfection into the cells and monitoring the expression of target proteins with the method of Western blotting. The target proteins were from families of cyclins and cyclin-dependent kinases, which play an important role in cell cycle regulation. The mRNAs for 8 types of molecules have been prepared, including non-mutated forms of cyclins D1, D2, and D3, and cyclin-dependent kinase 6, as well were synthesized mutated versions of cyclins D1 T286AT288A, D2 T280A, D3 T283A and cyclin-dependent kinase 4 R24C. As for cyclins, there was a substitution of aminoacid threonine for alanine. This substitution could cause slowdown degradation of proteins in comparision with those existing in natural form. Regarding the cyclin- dependent kinase 4 the exchange of aminoacid arginine for cysteine causes its bigger stability in vivo. The prepared mRNA which encodes mentioned regulators of cell cycle was transfected into isolated...
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