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Barley Proteomic Studies Related to Beer Production
Benkovská, Dagmar ; Márová, Ivana (oponent) ; Ehrenbergerová, Jaroslava (oponent) ; Zdráhal, Zbyněk (oponent) ; Bobáľová, Janette (vedoucí práce)
This work is focused on barley proteomic studies in relation to the beer production. Barley belongs between the most important crops in the world and its greatest use is for malting purposes, most commonly for the brewing industry. Studies of barley proteins during malting and brewing provide information about changes in protein composition or their post translational modifications. Since the protein composition and their modifications are essential for the quality of malt and beer, barley proteomic studies have a potential to improve the malting and brewing process. The main goal of this thesis is to investigate barley water-soluble proteins and their changes that occur during the malting and brewing process. The differences in protein composition were investigated using gel electrophoresis, reversed phase and size exclusion liquid chromatography, and MALDI-TOF mass spectrometry. The amount of some proteins is increasing and some new proteins are created in the germinated grain during the malting process. Contrary, many proteins are decomposed during the brewing process due to the high temperature and enzymatic activity of some proteases. Only some proteins belonging to the family of pathogenesis related proteins resist these harsh conditions and pass into the beer where they can influence several important quality properties. Furthermore, various barley varieties and their differences were investigated. Varieties allowed for the production of certified Czech beer were compared to one variety with well proven malting properties and one non-malting barley variety. In addition, alcohol soluble barley proteins and their changes during the malting process were investigated as well. A special attention was paid to selected post-translational modifications of proteins, namely glycosylations. Non-enzymatically glycosylated barley proteins (or glycated proteins) are formed during the malting process considering the large amount of glucose released from the starch degradation, and influence the protein stability as well as the beer quality, especially foaming properties. Enzymatic N-glycosylation represents the most frequently studied post translational modification in plants because glycoproteins play a key role in various biological functions. Since glycoproteins are often present in a small amount, their enrichment from a complex mixture is required for their analysis. Lectin concanavalin A affinity chromatography was used for barley glycoproteins investigation. Moreover, the analysis of the carbohydrate part of glycoproteins was optimized. This doctoral thesis brings important information about barley proteins, their modifications and analysis that are useful for further studies.
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Využití proteomiky pro monitorování potenciálních biomarkerů rakoviny prsu
Garcia N Dua, Felicita ; Ing.Anna Kubesová, Ph.D. (oponent) ; Flodrová, Dana (vedoucí práce)
Nádor prsu je jedním z nejčastějších maligních karcinomů u žen a prognóza úzce souvisí s tím, v jakém stádiu kancerogeneze je proces zachycen. V posledních letech je pozornost soustřeďována na hledání nových látek, jež by snížily negativní dopady, které s sebou nese chemoterapie. Bylo zjištěno, že jednou z efektivních látek v boji s karcinomem jsou retinoidy, které mají schopnost inhibovat nádory prsní žlázy v časných stádiích nemoci. Tato práce se zabývá proteomickými studiemi buněk karcinomu prsu a porovnáváním obsahu bílkovin obsažených v karcinomových buňkách, které byly podrobeny retinoidové léčbě. Úspěšně bylo využito několika separačních metod jako 1D a 2D elektroforéza, chromatografie na reverzní fázi a gelová permeační chromatografie. K tomu byly vzorky ještě podrobeny enzymatickému štěpení vybraných proteinů a jejich identifikaci pomocí MALDI-TOF MS. 2D gelová elektroforéza odhalila výskyt skupiny proteinů řadících se mezi molekulární chaperony, které jsou dnes považovány za významné specifické biomarkery nejen karcinomu prsu.
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Protein fractionation and relative quantitation using PF 2D and ITRAQ for biomarker quest
Gadher, S. J. ; Skalníková, Helena ; Halada, Petr ; Řehulka, Pavel ; Chmelík, Josef ; Kovářová, Hana
ProteomeLab PF 2D System - Protein Fractionation in 2 Dimensions (Beckman Coulter, Fullerton, CA, USA) has been developed to fractionate complex protein mixtures by chromatofocusing in the first dimension followed by high-resolution non-porous silica reversed phase chromatography (RP LC) in the second dimension. Despite the high-resolution power of ProteomeLab PF 2D, UV-based quantitation could be compromised due to possible co-elution of several proteins into one fraction. Hence, we present an optimized protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and MALDI-TOF/TOF mass spectrometry to obtain quantitative data from peptides derived by tryptic digestions of intact proteins fractionated by ProteomeLab PF 2D technique. To demonstrate the feasibility of such an approach, protein expression patterns obtained from the ProteomeLab PF 2D fractionation of human T-lymphoblastic leukemia CEM cell line were utilised.
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Proteomika CDK inhibice v nádorových buňkách
Kovářová, Hana ; Skalníková, Helena ; Halada, Petr ; Strnad, M. ; Hajdúch, M.
Využíváme komplexního proteomického přístupu pro studium biochemického principu protinádorového účinku syntetických inhibitorů cyklin dependentních kináz (CDKI) a pro hledání nových proteinů asociovaných s těmito biologickými účinky. Nádorové buňky dvou buněčných linií reprezentujících hematologickou malignitu a solidní tumor kultivujeme v přítomnosti nebo absenci CDKI Boheminu. Celulární proteiny těchto linií extrahujeme a frakcionujeme pomocí klasické dvourozměrné gelové chromatografie a take pomocí dvourozměrné kapalinové chromatografie (systém PF2D) Získané proteinové mapy počítačově vyhodnocujeme a diferenční proteiny identifikujeme hmotnostní spektrometrií. Tyto proteiny jsou kandidátními biomarkery protinádorové odpovědi na inhibici cyklin dependentních kináz.
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