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Proteomic identification of nuts in festive cookies
Kadeřábková, Lucie ; Kučková, Štěpánka (advisor) ; Kolář, Karel (referee)
This diploma thesis is devoted to the proteomic identification of selected nuts in festive cookies. Two types of mass spectrometry were used for the nut identification; their outcomes allow for comparison and completeness of the data. The theoretical part provides a comprehensive overview of the nutritional values of nuts and the issues of food fraud and authenticity. It concludes with a didactic view and potential applications in chemistry lessons and other school subjects. The experimental part analysed eleven different nuts, submitted to high temperatures, which simulated baking and seven different types of festive cookies (six mass-produced cookies and one type of cookie with priorly unknown content of nuts). Mass spectrometry MALDI-TOF was used with subsequent evaluation using relational database system PostgreSQL to obtain characteristic m/z values for each of the eleven heat-subjected nuts. These values were then compared to the m/z values of the festive cookies to identify declared nuts and nuts used in the "unknown" sample. This identification was proved with various success rates in four of six mass-produced samples. The success rate was derived from the number of unique m/z values found in each of the nuts. The "unknown" sample was correctly identified based on the presence of one third...
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The development of new radical cross-linkers for structural characterization of proteins
Karpíšek, Michael ; Kukačka, Zdeněk (advisor) ; Chmelík, Josef (referee)
Proteins are important biomolecules because they carry out many essential functions. Since the structure of these biomolecules is usually closely linked to their function, there are numerous techniques to determine their three-dimensional structure. Exploiting the advantages of mass spectrometry, chemical cross-linking coupled to mass spectrometry is one of the methods that is used for structural characterization of studied molecule. Currently there is no reagent that targets and links all of the aromatic proteinogenic residues found in the sequence of proteins. In this work, we verified the reactivity of newly developed cross-linker towards aromatic residues and sulfhydryl group of cystein. This new reagent is based on the so-called Togni reagents that serve as trifluoralkylation agents. First, we monitored the impact of several different conditions on the composition of the reaction products using apomyoglobin as a model protein and electrosprey ionization as the method of choice. Subsequently we employed bottom up approach to find out which residues were modified. For this purpose we chose apomyoglobin and two other proteins that contain cysteine in its reduced form - RhoA and HSC70. Based on obtained results, we could confirm the ability of the new cross-linker to modify both aromatic residues...
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Studying protein structure and interactions by structural mass spectrometry
Portašiková, Jasmína Mária ; Man, Petr (advisor) ; Vrbacký, Marek (referee)
Transmembrane channels and transporters of the ClC protein family are present across all living organisms. They are found on the cytoplasmic and lysosomal membranes of the cells, where they participate in maintaining ion homeostasis. When dysfuncional, they lead to serious health complications. To develop treatment for these diseases, it is essential to describe transport mechanism of ClC proteins. The antiporter ClC-ec1 from E.coli is used as a model protein for the entire ClC protein family. This homodimeric protein, which transports one proton against two chloride ions, has a separate transport path in each monomer. Based on the crystal structure, it is believed that during transport the protein alternates between outward and inward-facing conformations. Conversion to the outward-facing conformation of the protein is accompanied by the protonation of three glutamates located in the transport path. To study these conditions, a QQQ mutant was designed that has these glutamates replaced by glutamines. Until now, the study of the transport mechanism of ClC-ec1 has mainly relied on studies based on X-ray crystallography. Crystallography provided static images, which did not contain sufficient information about protein dynamics. Therefore, to study transport mechanism of ClC-ec1, we chose a dynamic...
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Proteomic resolution of cereals
Nyškovská, Kristýna ; Kučková, Štěpánka (advisor) ; Kolář, Karel (referee)
This bachelor thesis deals with the proteomic differentiation of cereals (amaranth, sown barley, sown maize, qinoa, grain sorghum, sown oats, sown wheat, sown buckwheat, sown millet, sown rice, spelt, sown rye). Cereals are abundant in the human diet and are one of the main sources of protein and carbohydrates. The aim was to determine whether it is possible to distinguish between different types of cereals and to compare the difference between cereals and pseudocereals based on the protein composition of their grains using the mass spectrometric method MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization - Time of Flight. The theoretical part deals with the introduction of cereals, pseudocereals and their nutritional values, application and analytical method MALDI-TOF MS. The experimental part deals with the collection of different cereal species and their subsequent analysis. The samples were mechanically processed and digested using the enzyme trypsin. The cleaved peptides were measured by MALDI-TOF MS and the obtained spectra were subsequently processed using mMass and PostgreSQL (version 14.2). In this way, specific m/z values were determined that are characteristic of each cereal analyzed. From these values, a table was compiled that summarizes these characteristic values. Which allows the...
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Possibilities of pathogen identification using MALDI-TOF mass spectrometry
Fusková, Lucie ; Strouhalová,, Dana (referee) ; Šalplachta, Jiří (advisor)
This bachelor thesis focuses on the utilization of MALDI-TOF MS technique for the identification of plant pathogens, specifically targeting molds of the genus Fusarium affecting cereals. Molds pose a serious threat to agriculture and the food industry. Current identification methods are time-consuming and often fail to provide precise results. Mass spectrometry offers a rapid and reliable alternative. Identification via MALDI is characterized by its high accuracy, sensitivity, and speed compared to commonly used methods. The thesis addresses the optimization of the method using cultured fungal strains and the identification of molds in infected cereals. The results of the study demonstrate the potential of utilizing MALDI technique for the identification of plant pathogens.
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Analysis of perfluorinated alkyl compounds in environmental matrices
Šimáková, Anna Marie ; Řezáčová, Veronika (referee) ; Mravcová, Ludmila (advisor)
The bachelor thesis deals with the still topical issue of perfluorinated and polyfluorinated substances in the aquatic environment. In the theoretical part, the production and use of PFASs, which has led to the spread of these pollutants into the environment, is first addressed. Then, their behaviour in the environment is presented, which, due to their properties, results in their persistence in the environment, their ability to accumulate in living organisms and, in the case of decomposition, the possibility of even worse impacts. Exposure can result in carcinogens, hormonal changes and various types of toxicity in living organisms. The theoretical part concludes with methods for possible extraction from different matrices and detection by a suitable analytical method. The experimental part deals with the optimization of parameters for the method of determination and extraction of perfluorinated and polyfluorinated substances from water. All parameters were optimized to achieve the highest possible recoveries with acceptable repeatability and to achieve the lowest possible limits of detection and quantification.
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Determination of pesticides in solid environmental matrices
Řepková, Michaela ; Komendová, Renata (referee) ; Mravcová, Ludmila (advisor)
The bachelor thesis deals with the still pressing issue of pesticides in the soil environment. The theoretical part deals with the entry of these pesticide substances into the environment, i.e. their application in various forms to agricultural soil, where they are retained in the soil based on certain factors and subsequently transported from the soil to other components of the environment. As pesticide residues are also present in other parts of the environment, they represent a global problem because of the potential environmental and health risks not only for the human population. Furthermore, the theoretical part deals with the appropriate method for the determination of these pesticide substances in the soil matrix, both a suitable extraction method and a suitable analytical technique. The experimental part primarily focused on the actual determination of these persistent organic pollutants. Pesticides from soil were extracted by one of the most popular extraction methods at present, namely the QuEChERS method. This method has been thoroughly optimised to achieve the lowest possible detection/quantification limits, the highest possible recoveries and good repeatability. The LC-MS/MS method was chosen as the appropriate analytical method for the determination of pesticides in soil extracts.
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Optimization of methods for the analysis of pharmaceuticals residues in different crops
Súkeníková, Kamila ; Zlámalová Gargošová, Helena (referee) ; Mravcová, Ludmila (advisor)
Aim of this thesis is to optimize the extraction method for extracting pharmaceuticals from various types of matrices. Presence of pharmaceuticals in the environment represent an increasing problem, even though they are only found in low concentrations. Therefore, great emphasis is placed on their analysis. Since the concentration of pharmaceuticals is often low and their chemical diversity is large, their analysis presents a significant challenge. For this reason, this thesis is focused on optimizing the extraction method from plant matrices. This optimization is carried out in order to obtain the best possible extraction method, and thus ensuring the highest possible yield of pharmaceuticals from radish, carrot, and mustard matrices. The optimization of the extraction method involved finding the parameters for individual steps of the QuEChERS extraction method that showed the best average yield for all pharmaceuticals.
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