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Hydrophilic Interaction Liquid Chromatography in the Analysis of Intact Glycopeptides
Molnárová, Katarína
The analysis of glycoproteins represents a significant challenge in glycoproteomics, primarily due to the macro- and microheterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is a convenient alternative to reversed-phase chromatography, commonly used in glycoproteomic analysis. This dissertation thesis discusses the potential of HILIC in glycoproteomic analysis, ranging from the separation of glycopeptides on polar stationary phases to the use of HILIC in sample preparation processes. First, the effect of acetonitrile concentration on glycopeptide precipitation was investigated, depending on the type of glycan attached. Subsequently, three commercially available stationary phases were tested: a column containing a silica gel modified with five hydroxyl groups, an amide stationary phase, and a zwitterionic stationary phase. Their efficiency in separating glycopeptide isomers, differing only in branching and/or linkage position, was compared. Further research was devoted to the separation of human immunoglobulin G glycopeptides using relatively new columns that have not yet been characterized in glycoproteomic analysis. These columns, provided by Advanced Chromatography Technologies, included unmodified silica gel (HILIC-A), aminopropyl- modified sorbent...
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Hydrophilic Interaction Liquid Chromatography in the Analysis of Intact Glycopeptides
Molnárová, Katarína ; Kozlík, Petr (advisor) ; Coufal, Pavel (referee) ; Cvačka, Josef (referee)
The analysis of glycoproteins represents a significant challenge in glycoproteomics, primarily due to the macro- and microheterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is a convenient alternative to reversed-phase chromatography, commonly used in glycoproteomic analysis. This dissertation thesis discusses the potential of HILIC in glycoproteomic analysis, ranging from the separation of glycopeptides on polar stationary phases to the use of HILIC in sample preparation processes. First, the effect of acetonitrile concentration on glycopeptide precipitation was investigated, depending on the type of glycan attached. Subsequently, three commercially available stationary phases were tested: a column containing a silica gel modified with five hydroxyl groups, an amide stationary phase, and a zwitterionic stationary phase. Their efficiency in separating glycopeptide isomers, differing only in branching and/or linkage position, was compared. Further research was devoted to the separation of human immunoglobulin G glycopeptides using relatively new columns that have not yet been characterized in glycoproteomic analysis. These columns, provided by Advanced Chromatography Technologies, included unmodified silica gel (HILIC-A), aminopropyl- modified sorbent...
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Determination of low-molecular-mass heparin using affinity capillary electrophoresis
Molnárová, Katarína ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
Unfractionated heparin, which is a widely used anticoagulant, is frequently replaced with low-molecular-mass species. They are used due to their more predictable anticoagulant effect with less bleeding complications and also they have prolonged anticoagulant effect. For monitoring of low-molecular-mass heparin levels, anti-factor Xa assay is used, which has some significant drawbacks. This work is dedicated to determination of low-molecular-mass heparin, namely Fraxiparine, using affinity capillary electrophoresis. Heparin is a polysaccharide which does not exhibit a significant UV absorption; therefore, its indirect detection method was used. Fraxiparine forms a stable complex with protamine. Protamine is an arginine-rich, positively charged peptide which is used to suppress heparin anticoagulant effect. Because protamine has a complex, not precisely defined structure, it was replaced by well-defined tetraarginine. The method uses phosphoric acid of 9 mmol L-1 concentration with addition of 0.1% (w/v) hydroxyethylcellulose as the background electrolyte. The samples are injected hydrodynamically into the capillary by a pressure of 5 kPa. First, the zone of Fraxiparine was injected, followed by the zone of tetraarginine (5 s). After that, 30 kV voltage was applied for 30 s. During this time the...
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Optimization of electrophoretic determination of protamine and insulin
Molnárová, Katarína ; Křížek, Tomáš (advisor) ; Hraníček, Jakub (referee)
This work deals with optimization of a method for separation and detection of protamine and insulin using capillary zone electrophoresis. The composition of background electrolyte, the solution pH and the injection method were optimized. Citric acid in a concentration range of 80 to 240 mmol L-1 and chloroacetic acid ranging from 50 to 150 mmol L-1 were tested sequentially. The optimized method uses a fused silica capillary with inner diameter of 50 μm. The total length of capillary is 50.0 cm, effective length is 8.5 cm. The injection of the sample is performed on the short end of the capillary. The method uses chloroacetic acid of 100 mmol L-1 concentration as the background electrolyte. Driving voltage is 20 kV. Sample is injected hydrodynamically with a pressure of 5 kPa for 3 s. The analytes are detected spectrophotometrically at wavelength of 200 nm. The method allows for determination in case of protamines in concentration range between 4 μg mL-1 and 300 μg mL-1 and insulin from 5 μg mL-1 to 300 μg mL-1 . The limits of detection are 1 μg mL-1 for protamine and 2 μg mL-1 for insulin. Repeatability of migration times and peak areas tested at 30 μg mL-1 and 200 μg mL-1 concentration levels using hydrodynamic injection showed values of relative standard deviation lower than 6 % suggesting...
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The male and female identity in contemporary society with focus on advertising
Molnárová, Katarína ; Hubinková, Zuzana (advisor) ; Kašparová, Eva (referee)
This thesis deals with male and female identity in contemporary society, with narrower focus on its presentation in television commercials. The aim of the work is the realization of content analysis, aiming at depicting the characters of men and women in television commercials. Theoretical part of the thesis consists of four chapters. The first chapter describes the general theoretical basis of this thesis. The second and third chapter deals with the specifics of male and female identity. The fourth chapter describes the research method of content analysis and presents the objectives and research hypotheses. The analytical part of the work examines the representation of men and women in television commercials at two levels - at the level of main characters, and at the level of commercials. The final part of the analytical part interprets the research results of analysis.
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