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Quest for protein biomarkers of neural stem cells differentiation
Skalníková, Helena ; Halada, Petr ; Vodička, Petr ; Motlík, Jan ; Horing, O. ; Jensen, O. N. ; Gadher, S. J. ; Pelech, S. ; Kovářová, Hana
Understanding neurogenesis and neural cell differentiation presents a unique challenge for treatment of nervous system disorders. To gain more insight about molecular mechanism of differentiation of neural cells, we applied different proteomic approaches using classical 2-DE followed by MS and antibody microarrays. Based on 2-DE, profile of constituent proteins of neural stem cells and their differentiated progenies was estabilished at first and then the protein species that are significantly up or down regulated during the differentiation were selected. Differentiation of neural cells was accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage and redox regulation. Immunoblot verified changes of hnRNP A1, hnRNP A2/B1, RafB, heme-oxygenasy 2, GRK2 proteins and alphaB-crystallin (S45), CDK1/2 (Y15), PKC mu (S738+S742), proline-rich Akt substrate (T246) phosphorylations during differentiation.
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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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