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Using proteomics for screening of potential breast cancer biomarkers
Garcia N Dua, Felicita ; Ing.Anna Kubesová, Ph.D. (referee) ; Flodrová, Dana (advisor)
Breast cancer is one of the most common malignant cancers and the prognosis is closely related to the stage of carcinogenesis process in which it was captured. Lately, attention was focused on the research of new compounds that would reduce the negative impacts of chemotherapy. It was found that retinoids are effective compounds in the fight against cancer. Retinoids have the ability to inhibit tumors of the mammary gland in an early stage of disease. This work deals with the proteomic studies of breast cancer cells and comparing the proteins contained in the cancer cells which were subjected to retinoid therapy. The differences in protein composition were investigated using gel electrophoresis, reversed phase and size exclusion liquid chromatography. The identification and characterization of selected proteins was carried out by MALDI-TOF MS. The use of two-dimensional gel electrophoresis led to the identification of molecular chaperons, which are considered to be important biomarkers not only of breast cancer.
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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.
In order to improve our understanding of the biochemical basis of the anti-cancer activity of olomoucine-derived synthetic cyclin-dependent kinase inhibitors (CDKIs) and to search for novel proteins associated with these biological effects we applied complex proteomic approaches. To analyse cellular responses to the CDKI we used two cancer models: the CEM T-lymphoblastic leukemia cell line representing hematological malignancy, and the A549 lung adenocarcinoma cell line as a solid tumor model. Cancer cells of these lines were cultured in both the presence and absence (controls) of the CDKI, bohemine (BOH). Cellular proteins of both of these lines were then extracted and fractionated using conventional two-dimensional gel electrophoresis (2-DE), and for the CEM T-lymphoblastic leukemia cell line we also used a 2-D liquid phase fractionation system ProteomeLab PF 2D ( Beckman Coulter). Computer-assisted data analysis of the resulting 2-D protein expression maps was applied to determine the similarity/dissimilarity of the maps and to select characteristic protein spots or bands based on the quantitative differences between BOH-treated and control cells. Many of these differentially expressed proteins were identified by mass spectrometry, since they represent candidate biomarkers of cancer cell responses to CDK inhibition and cellular pathways that are relevant to the anti-cancer activity of the CDKIs. Subsequently, we focused directly on these proteins in confirmatory studies using various techniques (including quantitative immunoblotting, immunocytochemistry and functional activity analyses) to demonstrate the validity of the proteomic results and extend our knowledge of the CDKI effects.
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