BD(TM) Free Flow Electrophoresis (FFE) System for protein and cell organelle purifications provides a tool to reduce sample complexity and enrich low abundant proteins with high resolution and reproducibility. A preliminary study was carried out to verify the possibility of direct coupling between FFE and MALDI-TOF. A first set of buffers, optimized by BD for MALDI compatibility, was used to prepare solutions of four different biomolecular systems (peptides mixture (PepMix), Ribonuclease B, proteins mixture (ProtMix) and Cytochrome C) at four different concentrations (20 µM, 2 µM, 0.2 µM, 0.02 µM). These solutions, as such or purified on ZipTip C18 cartridges, were analysed through MALDI-TOF and results compared with those obtained for the same substances,in the same concentrations, dissolved in CH3CN/TFA 0.1% 1/1, to evaluate the effect of buffers in MALDI analyses. When dissolved in CH3CN/TFA 0.1% 1/1, PepMix and ProtMix were detectable up to 0.2 µM concentration while Rnase B and Cyt C were detectable up to 2 µM concentration. In buffers 2 µM was the limit concentration under which it was impossible to detect most of our biomolecular systems. Therefore these buffers proved to be MALDI compatible, having only a weak effect in decreasing ionization efficiency. The use of ZipTip C18 cartridges produced better results only in the case of Rnase B. In a second experiment a mixture of three peptides plus Cytochrome C and a mixture of four proteins were separated by FFE using a further set of buffers optimized for MALDI analyses. Each well obtained from the two FFE separations was analysed by MALDI-TOF analysis. FFE/MALDI-TOF coupling was found to be an efficient system for the separation and analysis of the proteins mixtures whereas the protocol for the peptides mix plus Cytochrome C still requires further optimization. Finally, a real sample consisting of a homogenate silk month antennae was purified with the FFE System using the first set of buffers supplied by BD. Both satisfactory separation and good response in MALDI analyses were obtained.
Direct coupling of FFE protein separation and MALDI-TOF analysis
E Michelucci;
2008
Abstract
BD(TM) Free Flow Electrophoresis (FFE) System for protein and cell organelle purifications provides a tool to reduce sample complexity and enrich low abundant proteins with high resolution and reproducibility. A preliminary study was carried out to verify the possibility of direct coupling between FFE and MALDI-TOF. A first set of buffers, optimized by BD for MALDI compatibility, was used to prepare solutions of four different biomolecular systems (peptides mixture (PepMix), Ribonuclease B, proteins mixture (ProtMix) and Cytochrome C) at four different concentrations (20 µM, 2 µM, 0.2 µM, 0.02 µM). These solutions, as such or purified on ZipTip C18 cartridges, were analysed through MALDI-TOF and results compared with those obtained for the same substances,in the same concentrations, dissolved in CH3CN/TFA 0.1% 1/1, to evaluate the effect of buffers in MALDI analyses. When dissolved in CH3CN/TFA 0.1% 1/1, PepMix and ProtMix were detectable up to 0.2 µM concentration while Rnase B and Cyt C were detectable up to 2 µM concentration. In buffers 2 µM was the limit concentration under which it was impossible to detect most of our biomolecular systems. Therefore these buffers proved to be MALDI compatible, having only a weak effect in decreasing ionization efficiency. The use of ZipTip C18 cartridges produced better results only in the case of Rnase B. In a second experiment a mixture of three peptides plus Cytochrome C and a mixture of four proteins were separated by FFE using a further set of buffers optimized for MALDI analyses. Each well obtained from the two FFE separations was analysed by MALDI-TOF analysis. FFE/MALDI-TOF coupling was found to be an efficient system for the separation and analysis of the proteins mixtures whereas the protocol for the peptides mix plus Cytochrome C still requires further optimization. Finally, a real sample consisting of a homogenate silk month antennae was purified with the FFE System using the first set of buffers supplied by BD. Both satisfactory separation and good response in MALDI analyses were obtained.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
