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Application of acid proteases from nepenthes in hydrogen/deuterium exchange.
Darebná, Petra ; Man, Petr (advisor) ; Stráňava, Martin (referee)
Application of acid proteases from Nepenthes in hydrogen/deuterium exchange Petra Darebná (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Nepenthes are mostly found in Borneo and Sumatra. They are one of a few carnivorous plants which produce its own proteolytic enzymes (nepenthesin I and nepenthesin II), which provide an alternative source of nitrogen and other nutrients in case that these plants grow in a soil which lacks such nutrients. These aspartate proteases are capable of proteolysis at a very low pH of a digestive gastrovascular cavity fluid after catching insects and in a cooperation with other proteins participating in digestion process. Processing of a digestive fluid, isolated from a digestive gastrovascular cavities of carnivorous plants of the Nepenthes genus and its possible application as a tool in a protein study using hydrogen/deuterium exchange were done in this thesis. Isolates of the digestive fluids were purified from coarse-grained impurities by centrifugation, activated by acidification and concentrated by ultrafiltration. The amount of proteins and their protein profile were monitored and an activity of acidic proteases was determined by enzymatic assay. Consequently, using LC-MS/MS and model proteins, the cleavage preferences...
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Characterization of hapt oglobin glycoforms in human serum
Darebná, Petra ; Pompach, Petr (advisor) ; Ptáčková, Renata (referee)
Characterization of haptoglobin glycoforms in human serum Petra Darebná (Katedra biochemie, Přírodovědecká fakulta, Univerzita Karlova v Praze, Česká republika) Changes in glycosylation of proteins are associated with several types of cancer, including hepatocelular carcinoma and colorectal carcinoma. This project deals with data independent analysis using ion cyclotron resonance with Fourier transform and tandem mass spectrometry with liquid chromatogramy and multiple reaction monitoring to quantify these changes in hepatocelular cancinoma and colorectal carcinoma with liver metastases. In the first part of the project the haptoglobin was enriched from pooled serum samples of pacients with hepatocellular carcinoma, colorectal cancer and colorectal carcinoma with metastases using hemoglobin immobilized on CNBr-activated Sepharose 4B and then purified by high pressure liquid chromatography. Individual haptoglobin glycopeptides were analyzed using ion cyclotron resonance with Fourier transform. In the second part of the project we analyzed changes in glycosylation depending on diferent tumor diseases in partially depleted serum of individual patients using ethanol precipitation. Individual fucosylated glycoforms of N-glycopeptides of serum proteins were compared with their nonfucosylated forms. In...
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Characterization of hapt oglobin glycoforms in human serum
Darebná, Petra ; Pompach, Petr (advisor) ; Ptáčková, Renata (referee)
Characterization of haptoglobin glycoforms in human serum Petra Darebná (Katedra biochemie, Přírodovědecká fakulta, Univerzita Karlova v Praze, Česká republika) Changes in glycosylation of proteins are associated with several types of cancer, including hepatocelular carcinoma and colorectal carcinoma. This project deals with data independent analysis using ion cyclotron resonance with Fourier transform and tandem mass spectrometry with liquid chromatogramy and multiple reaction monitoring to quantify these changes in hepatocelular cancinoma and colorectal carcinoma with liver metastases. In the first part of the project the haptoglobin was enriched from pooled serum samples of pacients with hepatocellular carcinoma, colorectal cancer and colorectal carcinoma with metastases using hemoglobin immobilized on CNBr-activated Sepharose 4B and then purified by high pressure liquid chromatography. Individual haptoglobin glycopeptides were analyzed using ion cyclotron resonance with Fourier transform. In the second part of the project we analyzed changes in glycosylation depending on diferent tumor diseases in partially depleted serum of individual patients using ethanol precipitation. Individual fucosylated glycoforms of N-glycopeptides of serum proteins were compared with their nonfucosylated forms. In...
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Application of acid proteases from nepenthes in hydrogen/deuterium exchange.
Darebná, Petra ; Man, Petr (advisor) ; Stráňava, Martin (referee)
Application of acid proteases from Nepenthes in hydrogen/deuterium exchange Petra Darebná (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Nepenthes are mostly found in Borneo and Sumatra. They are one of a few carnivorous plants which produce its own proteolytic enzymes (nepenthesin I and nepenthesin II), which provide an alternative source of nitrogen and other nutrients in case that these plants grow in a soil which lacks such nutrients. These aspartate proteases are capable of proteolysis at a very low pH of a digestive gastrovascular cavity fluid after catching insects and in a cooperation with other proteins participating in digestion process. Processing of a digestive fluid, isolated from a digestive gastrovascular cavities of carnivorous plants of the Nepenthes genus and its possible application as a tool in a protein study using hydrogen/deuterium exchange were done in this thesis. Isolates of the digestive fluids were purified from coarse-grained impurities by centrifugation, activated by acidification and concentrated by ultrafiltration. The amount of proteins and their protein profile were monitored and an activity of acidic proteases was determined by enzymatic assay. Consequently, using LC-MS/MS and model proteins, the cleavage preferences...
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