In Taberna Quando Sumus: a drunkard's cakewalk though proteomics

Comunicaciones a congreso

Proteomic studies of marine organism

Comunicaciones a congreso

Proteomic signature of non-alcoholic beverages via combinatorial peptide ligand libraries

Comunicaciones a congreso

Performance of a series of novel N-substituted acrylamides in capillary electrophoresis of DNA fragments

DNA separations by capillaryelectrophoresis in viscous solutions of novel polymers, made with \u3a9-hydroxyl, N-substitutedacrylamides (notably N-acryloyl amino propanol, AAP and N-acryloyl amino butanol, AAB) are evaluated. Whereas in standard poly(acrylamide), at 6% concentration, the theoretical plate number (N) does not exceed 500 000, in 6% poly(AAP) N reaches 922 000 and in 6% poly(AAB) N values as high as 1 200 000 are obtained. Also, copolymers of AAP and AAB give N values in excess of 1 million plates. The two novel monomers (AAP and AAB) remain extremely stable during alkaline hydrolysis and display very good hydrophilicity, while being devoid of the noxious habit of auto-polymerization and auto-reticulation exhibited by the previous monomer of this series (N-acryloyl amino ethoxy ethanol). The reasons for such a good performance of the \u3a9-substituted acrylamide derivatives could be that their polymers may form hydrogen bonds via their distal -OH group during DNA separation

Solubilization of Proteins in 2DE: An Outline

Protein solubilization for two-dimensional electrophoresis (2DE) has to break molecular interactions to separate the biological contents of the material of interest into isolated and intact polypeptides. This must be carried out in conditions compatible with the first dimension of 2DE, namely isoelectric focusing. In addition, the extraction process must enable easy removal of any nonprotein component interfering with the isoelectric focusing. The constraints brought in this process by the peculiar features of isoelectric focusing are discussed, as well as their consequences in terms of possible solutions and limits for the solubilization process

Binding of polyanions to carrier ampholytes in isoelectric focusing

The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate

Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid.

Capillary 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

Isoelectric focusing of heparin. Evidence for complexing with carrier ampholytes

The basis for heparin fractionation into 21 components by isoelectric focusing has been shown to be a strong interaction between the polysaccharide and different amphoteric species in the Ampholine mixture. This was demonstrated by altering the heparin/Ampholine ratio, by loading the sample either before focusing or to a prefocused gel slab and by re-running single heparin bands. The complexes exhibiting apparent pI values in the pH range 3.2--4.5 appear to be particularly stable, probably because an optimal amount of amino groups (4 to 5) in the Ampholine molecules are protonated. When stained with Toluidine blue, the heparin/Ampholine complexes precipitated in the gel exhibited different degrees of metachromasia, reflecting competition of the dye and individual components of Ampholine with respect to binding sites of heparin: at least three colours, violet, blue and indigo, are distinguishable. Ampholine, when added to a heparin . Toluidine blue complex in solution displaces the dye from the polysaccharide

Synthesis of buffers for generating immobilized pH gradients. II: Basic acrylamido buffers.

Six basic acrylamido buffers, for isoelectric focusing in immobilized pH gradients, are described: a strong titrant (pK greater than 12), QAE-acrylamide, and five weaker bases, namely: 2-morpholinoethyl acrylamide (pK 6.2), 3-morpholinopropyl acrylamide (pK 7.0), N,N-dimethyl aminoethyl acrylamide (pK 8.5), N,N-dimethyl aminopropyl acrylamide (pK 9.3) and N,N-diethyl aminopropyl acrylamide (pK 10.3). Their synthesis and general properties are described