1,721,056 research outputs found
Monitoring folding/unfolding trtansitions of proteins by capillary electrophoresis: measurement of DG and its variation along the pH scale
No full textGeneration of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid.
No full textCapillary zone electrophoresis in stationary, isoelectric buffers is a novel method for generating peptide maps of protein digests. The buffer system developed is composed of iminodiacetic acid (IDA), whose physico-chemical parameters were found - by theoretically modeling and experimental verification - to be: pI 2.23 (at 100 mM concentration), pK(1) = 1.73 and pK(2) = 2.73 (no attempts were made at measuring the pK of the primary amino group, since such a low pI value would be compatible with any pK value of the basic group, down to as low as pK 5.5). IDA is compatible with most hydro-organic solvents, including trifluoroethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 mM IDA, 10% TFE and 0.5% hydroxyethylcellulose (HEC) allows generation of peptide maps with high resolution, reduced transit times and no interaction of even large peptides with the wall. However, the best background electrolyte was found to be a solution of 50 mM IDA in 0.5% HEC and 6-8 M urea, one of the best solubilizers of proteins and peptides known. In this last electrolyte system, peptide maps of beta-casein digests (known to contain also very large peptides, up to 6000 Da) could be generated with excellent resolution and half the transit times as compared with the standard buffer adopted in peptide analysis (80 mM phosphate buffer, pH 2.0). IDA thus appears to be another valid isoelectric buffer system, operating in a different pH window (pH 2.33 in 50 mM IDA) as compared to the other amphotere previously adopted (50 mM Asp, pH 2.77) for the same kind of analysis
Omics | quantitative proteomics
No full textThe present review attempts to cover the vast array of methods that appeared in the last few years for performing quantitative proteome analysis. These methods are divided into two classes: those applicable to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (SDS electrophoresis) and those applicable to 2D chromatographic protocols. The first method, although being by and large the most popular approach, can offer differential display of paired samples with relatively few methods, the oldest one being based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PD-Quest, Z3 and Z4000, Phoretix and Progenesis. Recent developments comprise analysis performed on a single gel containing mixed samples differentially labeled, either with fluorophors (Cy3 and Cy5) or with d0/d3-acrylamide. Conversely, chromatographic approaches, which mostly rely on analysis not of intact proteins but of their tryptic digests, offer a panoply of differential labeling protocols, most of which rely on stable isotope tagging. Essentially all possible reactions have been described, such as those involving Lys, Asp, Glu and Cys residues, as well as a number of methods exploiting differential derivatization of amine and carboxyl groups generated during proteolysis. All such methods are described and evaluate
Purification of glycopeptide antibiotics by isoelectric focusing in multicompartment electrolyzers with Immobiline membranes.
No full text
Capillary electrophoresis of peptides and proteins in acidic, isoelectric buffers: recent developments
No full textThe use of isoelectric buffers in capillary zone electrophoresis is here reviewed. Such buffers allow delivery of very high voltage gradients (up to 1000 V/cm in relatively large bore capillaries, e.g. 75-100 mu m I.D.), permitting separations of the order of a few minutes and thus conserving (in fact favouring) very high resolution due to minimal, diffusion-driven, peak spreading. Isoelectric Asp (pl 2.77 at 50 mM concentration and 25 degrees C) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta globin chains. Also imino diacetic acid (pl 2.33 at 50 mM concentration) allows generation of high resolution peptide maps. Isoelectric Asp, in presence of 7 M urea and 0.5% hydroxyethyl cellulose (Mn = 27 000 Da) is also the preferred medium for fast separation and analysis of storage proteins in cereals, such as gliadins in soft and durum wheat and zeins in maize. A solution of 50 mM iminodiacetic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is effectively used as background electrolyte for fast separation of heme-free, denatured globin (alpha and beta) chains. In the presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to 'micellar electrokinetic chromatography'
Proteomic fingerprinting of apple fruit, juice, and cider via combinatorial peptide ligand libraries and MS analysis
No full textCombinatorial peptide ligand libraries coupled to MS was applied to extensively map the proteome of apple fruit, and to detect its presence in commercial apple juice and cider to evaluate their authenticity and genuineness. Using the Uniprot_Malus database, 96 proteins were detected in apples, among which 30 proteins were specifically captured via combinatorial peptide ligand libraries. Next, three proteins, previously recognized in fruits, were found in apple juice, which were involved in cellular metabolism of fruit maturation and in allergenic reactions. On the other hand, only one Malus allergen was identified in cider beads eluate, demonstrating that the industrial processes did not prevent any negative effects in sensitive subjects. Thus, the present study not only increases the knowledge of the apple proteome but also offers a reliable analytical method to assess quality and genuineness of commercial products, which could be also used to inform consumers about the presence of allergens
“Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A”
No full textA pancreatic adenocarcinoma cell line (Paca44) was treated with trichostatin-A (TSA), a potent inhibitor of histone deacetylases, in order to evaluate the effect of this drug on protein expression. Master maps of control and treated Paca44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 51 polypeptide chains, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry analysis enabled the identification of 22 of these spots. Among these proteins, of particular interest are the two downregulated proteins nucleophosmin and translationally controlled tumor protein, as well as the upregulated proteins programmed cell death protein 5 (also designated as TFAR19) and stathmin (oncoprotein 18). The modulation of these four proteins is consistent with our observation that TSA is able to inhibit cell growth of Paca44 by causing cell cycle arrest at the G2 phase and apoptotic cell death
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