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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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Influence of the laser power and analyte concentration on the results in MALDI-TOF MS of oligosaccharides
Čáslavský, Josef ; Chmelík, Josef
MALDI-TOF MS has been successfully applied for the analysis of a wide range of organic biopolymers including oligosaccharides. We focus our attention to the group of oligosaccharides occuring in beer and in malting and mashing products, which are mainly oligosaccharides containing from 3 to more than 30 glucose units in their molecules. We tried to evaluate various factors influencing the obtained results. We found that the shape of the spectrum depends significantly on the energy of ionising laser pulse and also on the amount of the analyte on the MALDI target. These effects were studied with standard mixtures as well as with real samples.
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