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New types of magnetic sorbents for phosphoprotein analysis
Emmerová, Tereza ; Kučerová, Zdenka (advisor) ; Ryšlavá, Helena (referee)
The method for the study of protein phoshorylation sites was elaborated. This method is based on the IMAC separation of phosphopeptides from protein proteolytic digests using new magnetic sorbents and on their subsequent identification by mass spectrometry (MS). Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) particles prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) with immobilized Fe(III) and Ga(III) ions were employed for the enrichment of phosphopeptides from the proteolytic digests of two model proteins, porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from both proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. For the immunochemical separation of phosphoproteins, protein fraction containing antibodies was obtained from egg yolk of hens immunized with O-phosphoryl-L-serine conjugated to key limpet hemocyanin. Antibodies were purified using affinity chromatography on immobilized α-casein and their presence was proven by MS. Specificity of the obtained antibodies was examined using ELISA tests. Obtained results showed, that specificity...
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Peptide inhibitors immobilized on magnetic particles and Sepharose used for separation of stomach aspartate proteinases
Rajčanová, Michaela ; Kučerová, Zdenka (advisor) ; Fusek, Martin (referee) ; Pacáková, Věra (referee)
IN ENGLISH Human gastric juice contains mainly aspartate proteinases: pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens and they are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. A subject of this thesis is a part of a long-term investigation that focuses on the elaboration of methods for separation gastric aspartate proteainases that would be suitable for diagnostic purposes. The preparation of new type ligands was a concrete subject of PhD. thesis that after their immobilization they can enable the separation of aspartate proteinases. Four heptapeptides containing D-leucinyl residue were synthetized (Val-D-Leu-Pro-Phe-Phe-Val- D-Leu, Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu and Val-D- Leu-Pro-Phe-Tyr-Val-D-Leu. The prepared heptapeptides immobilized on agarose magnetic particles were used for the study of their interaction with porcine pepsin A and rat pepsin C. While porcine pepsin A was adsorbed to all heptapeptides immobilized to magnetic particles, rat pepsin C was not retarded. Similar results were obtained using heptapeptides immobilized to Sepharose. The situation was more complicated...
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Development of Methods for High-Throughput Enrichment of Glycoproteins and Glycopeptides Employing Multiple Lectin Affinity Chromatography/Tandem Mass Spectrometry
Maděra, Milan ; Pacáková, Věra (advisor) ; Feltl, Ladislav (referee) ; Foret, František (referee) ; Kučerová, Zdenka (referee)
5 CONCLUSIONS This thesis surrrnrarizes the development of a multimethodological analýical approach employing microcolumn lectin affi1t1,9fuom1tography coupled to highir".otu,to; separation and dete9tion techniques, in order to facilitate the enrichmení of glyco-proteins aná giycopeptioes originating from small quantities of real samples. This is u"toauy a sipificant-ítep towara satis$'ing the demands of contemporary glycoproteomics, which rely orirurt *a small scale analyses, allowing the identification of low abundant protein components' The overď conclusions and contribution to the contemporaÍy scierrce are drawn in tbree followine chaoters. 5.1 combining Lectin Microcolumns with High-Resolution separation Techniques for Enrichment of Glycoproteins and Glycopeptides This section describes coupling small-scďe lectin aÍfinity chromatograpby on-line to high- resolution separation and detection techniques, with the utiliý of a microcolumn loaded with a lectin immobilized onto macroporous silica. Experimental results, involving optimization of coupling procedure, fabrication of lectin microcolumns, verification of tteir Uin&ng efficiency and their possibility of interfacing on-line with nano LC-MS/]víS, me summarizžd into thé following conclusions; o optimized coupling procedure involved resuspending only 125...
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High-performance chromatographic methods for determination of proteins
Vařilová, Tereza ; Pacáková, Věra (advisor) ; Feltl, Ladislav (referee) ; Coufal, Pavel (referee) ; Kučerová, Zdenka (referee)
Conclusions This Thesis gives an overview of high-performance riquid chromatography in various separation modes for a characterization of selected proteins. various affinity stationary phases were prepared for analysis of pepsin, seminal plasma proteins and selected glycoproteins' choice of the affinity system suitable for a particular purpose is always a comprex probrem in which number of aspects must be considered. properties of the proteins to be separated and ligands used had to be taken into an account. The separation system should not influence the biorogicai activity ofproteins separated. From this point of view it is clear that for any each individuar system is necessary to find specific conditions for I) immobilizationof ligand, and II) separation conditions. Stationary phases with immob'ized 3,5-diiodo-L-tyrosine exhibited sufÍicient selectivity, proved to be useful for analysis ofporcine pepsin A and are appricable to practical sampres of human pepsin' Reversed-phase crs corumn is suitable for anarysis ofpepsin fragments obtained after a-chymotrypsin digestion. A study oť interaction of various forms of pepsin and pepsinogen are essentiar because of their high importance as crinicar diagnostic markers and as stomach enzyme. "lA IŤ Aťfinity chromatography with heparin immobilized to Toyopearl...
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Optimization of urinary exosome isolation for proteomic analysis in kidney disease diagnosis
Ulrychová, Lucie ; Přikryl, Petr (advisor) ; Kučerová, Zdenka (referee)
Extracellular vesicles (exosomes) are the subject of current nephrology proteomics research as they are considered as a promising source of potential biomarkers of kidney disease. This work is focused on discovery of the most appropriate procedure for the urinary exosomes isolation. We have compared already described methods, based on different physicochemical principles of isolation: hydrostatic filtration dialysis (HFD), differential ultracentrifugation, ultrafiltration through a 100 kDa filter, or sample precipitation with Total Exosome Isolation (from urine) kit. Characterization of individual isolated exosomal fractions was performed using SDS-PAGE method (presence of contaminating proteins), western blot analysis (detection of exosomal markers TSG101, alix), nanoparticle tracking analysis (NTA, vesicle size and concentration) or transmission electron microscopy (TEM, vesicles morphology). Due to the presence of contaminating proteins in urine samples, which could distort the results of subsequent proteomic assays, the conditions for the cleavage of undesirable proteins by proteinase K prior to their own isolation were optimized. It has been found that the best yield and purity of the isolated exosomal fractions were provided by a process combining HFD with differential ultracentrifugation...
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Peptide inhibitors immobilized on magnetic particles and Sepharose used for separation of stomach aspartate proteinases
Rajčanová, Michaela ; Kučerová, Zdenka (advisor) ; Fusek, Martin (referee) ; Pacáková, Věra (referee)
IN ENGLISH Human gastric juice contains mainly aspartate proteinases: pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens and they are activated to the corresponding pepsins in the acidic environment of the gastric lumen. The levels of pepsinogens in serum reflect the morphological and functional status of gastric mucosa. A subject of this thesis is a part of a long-term investigation that focuses on the elaboration of methods for separation gastric aspartate proteainases that would be suitable for diagnostic purposes. The preparation of new type ligands was a concrete subject of PhD. thesis that after their immobilization they can enable the separation of aspartate proteinases. Four heptapeptides containing D-leucinyl residue were synthetized (Val-D-Leu-Pro-Phe-Phe-Val- D-Leu, Val-D-Leu-Pro-Tyr-Phe-Val-D-Leu, Val-D-Leu-Pro-Tyr-Tyr-Val-D-Leu and Val-D- Leu-Pro-Phe-Tyr-Val-D-Leu. The prepared heptapeptides immobilized on agarose magnetic particles were used for the study of their interaction with porcine pepsin A and rat pepsin C. While porcine pepsin A was adsorbed to all heptapeptides immobilized to magnetic particles, rat pepsin C was not retarded. Similar results were obtained using heptapeptides immobilized to Sepharose. The situation was more complicated...
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Characterization of enzyme reactor with immobilized alkaline phosphatase
Plecitá, Denisa ; Kučerová, Zdenka (advisor) ; Miarková, Eva (referee)
Phosphorylation is one of the most common of all post-translational modifications of proteins and has been found in nearly all cellular processes. Abnormal phosphorylation is associated with many serious human diseases. One of the approaches used for the identification of protein phosphorylation sites is based on the application phosphatases and the comparison of MS analysis of samples before and after the sample treatment with the enzyme. The use of phosphatase immobilized to magnetic carriers is advantageous in comparison with the application of soluble enzyme: e.g. easy manipulation of samples, an increase of enzyme stability and a possibility of repeated use of immobilized enzyme. Investigation of properties of enzyme reactor - bovine alkaline phosphatase from intestinal mucosa immobilized to magnetic particles is a subject of this Bachelor Thesis. The enzyme was coupled to cellulose magnetic particles after activation with divinyl sulfone via the protein free amino groups. p-Nitrophenylphosphate was used as a substrate for the phosphatase activity determination. The effect of different conditions on the activity of soluble and immobilized forms of alkaline phosphatase was compared: the effect of pH and Mg2+ ions, storage stability and thermostability and possibility of repeated use of the...
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New types of magnetic sorbents for phosphoprotein analysis
Emmerová, Tereza ; Ryšlavá, Helena (referee) ; Kučerová, Zdenka (advisor)
The method for the study of protein phoshorylation sites was elaborated. This method is based on the IMAC separation of phosphopeptides from protein proteolytic digests using new magnetic sorbents and on their subsequent identification by mass spectrometry (MS). Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) particles prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) with immobilized Fe(III) and Ga(III) ions were employed for the enrichment of phosphopeptides from the proteolytic digests of two model proteins, porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from both proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. For the immunochemical separation of phosphoproteins, protein fraction containing antibodies was obtained from egg yolk of hens immunized with O-phosphoryl-L-serine conjugated to key limpet hemocyanin. Antibodies were purified using affinity chromatography on immobilized α-casein and their presence was proven by MS. Specificity of the obtained antibodies was examined using ELISA tests. Obtained results showed, that specificity...
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The Separation of Gastric Aspartic Proteases Using Affinity Chromatography
Frýdlová, Jana ; Kučerová, Zdenka (advisor) ; Barthová, Jana (referee) ; Jonáková, Věra (referee)
Human gastric juice contains mainly aspartic proteases - pepsin A and pepsin C. Both pepsins are produced by gastric mucosa as inactive pepsinogens (pepsinogen A and pepsinogen C) that differ in their physico-chemical and immunological properties. Both pepsinogens consist of molecular variants, isozymogens. Pepsinogens are activated to the corresponding pepsins in the acidic environment of the gastric lumen. (...) A subject of this Ph.D. thesis is a part of a long-term investigation that focuses on the elaboration of methods for the separation of gastric aspartic proteases that would be suitable for monitoring of their changes in mentioned diseases. This thesis was mainly focused on preparation of affinity sorbents suitable for separation of pepsins and pepsinogens. The choice of ligands was based on the substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine that is used to differentiate pepsin A and pepsin C. The following three affinity sorbents were prepared: iodinated L-tyrosine-Sepharose, 3,5-diiodo-L-tyrosine- Sepharose, and N-acetyl-L-phenylalanine-Sepharose. The basic characteristics of the prepared affinity sorbents were determined using the model enzyme (porcine pepsin A). The comparison of the chromatographic behavior of porcine pepsin A and its complex with pepstatine A showed that the enzyme...
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