Spot overlapping in two-dimensional polyacrylamide gel electrophoresis maps: relevance to proteomics

Proteomics requires a large-scale, simultaneous separation of proteins from a mixture, assessment of the relative abundance of these molecules, and identification and characterization of each component. In 2-D PAGE separations, the best method of choice for protein analysis, separation of all the proteins present in the sample is still far to be achieved and comigrating proteins in the same spot are in general present. A statistical estimation of the degree of spot overlapping present in a 2-D PAGE separation is here described: for different conditions of spot overcrowding in the map, the degree of overlapping can be quantified in terms of purity degree of each spot or percentage of proteins that will appear in the map as a single spot. A computer simulation approach is described: it is based on the protein separation pattern present in the experimental maps. The results thus obtained are compared to a theoretical model (statistical degree of peak overlapping model) based on random spot position. The described procedures were applied to an experimental reference map of human plasma. The severity of spot overlapping in 2-D PAGE maps is estimated and the influence of different experimental conditions (strip dimension, detector system performance, pI range) is discussed. These informations are useful to quantitatively estimate the degree of error associated with identification and quantitation of each protein and to set-up experimental conditions which will increase resolution and greatly decrease the probability of spot overlapping.     

A new deuterated alkylating agent for quantitative proteomics

Weakly basic molecules containing a double bond, such as 2- and 4-vinylpyridine, are able to react and selectively alkylate -SH groups in proteins, thus preventing their re-oxidation to disulphide bridges. In contrast to conventional alkylating agents such as iodoacetamide and non-charged acrylamide derivatives, such molecules achieve 100% alkylation of all -SH residues, even in complex proteins, without reacting with other functional groups. Their use is particularly effective in proteome analysis and more generally for analyzing proteins in which the -SH groups should be blocked. Additionally, the use of vinylpyridines, partially or totally deuterated and thus with a mass difference compared with their non-deuterated counterparts of 4-7 Da, allows studies of induction/repression of protein synthesis (quantitative proteomics).     

Regioselective one-pot synthesis of 9-alkyl-6-chloropyrido[3,2-e][1,2,4]triazolo[4,3-a]pyrazines. Reactivity of aliphatic and aromatic hydrazides

[reaction: see text] The one-pot synthesis of new 9-alkyl-6-chloropyrido[3,2-e][1,2,4]triazolo[4,3-a]pyrazines has been achieved. Hydrazides regioselectively reacted as nucleophiles with the 3-chloro substituent of 2,3-dichloropyrido[2,3-b]pyrazine. An intramolecular cyclization afforded the tricycle nonxanthine adenosine receptor antagonists.     

Potentiometric determination of boron in silicon with an ion-selective electrode

A potentiometric method is proposed for the determination or boron in silicon, based on dissolution of silicon by treatment with hydrofluoric acid and ammonium fluoride in the presence of hydrogen peroxide, and conversion of boron to fluoroborate ion. The fluoroborate activity is measured with the Orion fluoroborate-selective electrode. Some important points, such as the effect of various interfering ions and the hydrolysis of fluoroborate ion, are described in detail. The method is applied to the determination of boron in silicon containing at least 10 p.p.m. of boron, with a relative standard deviation of ±4%.     

A simplified model for the coupled transport of metal ions through hollow-fiber supported liquid membranes

This paper describes a simplified model for the carrier-facilitated transport of metal ions through hollow-fiber supported liquid membranes, HFSLM. The model leads to approximate and simple equations describing the concentration variations expected when an aqueous feed solution is flowing through the lumens of a HFSLM module. The equations incorporate simple and independently measurable parameters and apply to two situations: (a) a once-through mode, i.e., the feed solution passes only once through the module, and (b) a recycling mode, i.e., the feed solution is continuously recirculated through the module. The equations have been tested by measuring the transport of Cu²⁺ ions through microporous polypropylene hollow fibers containing a 0.3 F solution of bis(2-ethylhexyl)phosphoric acid in n-dodecane. HFSLM modules containing a variable number of fibers, fibers of different lengths and operated at different linear flow velocities have been used.     

Structural and vibrational properties of tetraoxaporphyrin dication, the oxygen analogue of porphyrin, and of isoelectronic diprotonated porphyrin

Structural calculations by means of the density functional method have been performed on tetraoxaporphyrin dication and on isoelectronic diprotonated porphyrin as well as on the sulfur and carbon analogues of porphyrin. A detailed study of the stable conformations of these compounds is reported starting with the most symmetrical conformations and lowering the symmetry along the vibrational coordinates with imaginary frequency. The calculated geometries are related to experimental structures available from X-ray diffraction studies. The Raman spectra of tetraoxaporphyrin dication exciting with micro-Raman instrumentation at 785 nm and of diprotonated porphyrin in near-resonance conditions with the Soret band have been measured. The correlation between frequencies calculated with the DF/B3-LYP/cc-pVDZ procedure for porphyrin, diprotonated porphyrin, and tetraoxaporphyrin dication has allowed for making a vibrational assignment for the latter two systems in excellent agreement with experiment using a single frequency scale factor.     

Micellar Solubilization of Biopolymers in Hydrocarbon Solvents III. Empirical Definition of an Acidity Scale in Reverse Micelles

This paper deals with the problem of defining, and measuring, the pH inside the water pool (which we define as pH_wp) of reverse micelles, i.e. micelles formed by surfactants dissolved in apolar solvents in the presence of minimal amounts of water. The conceptual and experimental difficulties are discussed, and it is argued that no absolute determination of pH_wp is possible, mostly because water in the water pools of reverse micelles is a new solvent, for which no standardization of acidity is available. The problem can be approached only on the basis of an empirical acidity scale. An empirical acidity scale for water pools in reverse micelles of bis (2-ethyl-hexyl) sodium sulfosuccinate (AOT) in isooctane has been defined by measuring the ³¹P-chemical shifts of phosphate buffers. The chemical shifts in bulk water were compared to those found in reverse micelles under the assumption that the pK of phosphate ion is the same in the two systems. It was found that in most cases there was little difference (less than 0.4 pH units) between pH_wp and the pH of the starting buffer in bulk water (which we define as pH_st). However, this difference between pH_wp and pH_st may become much larger in certain cases. The difference (pH_wp–pH_st) is measured under a variety of conditions, and this permits the determination of an operational acidity in the micelle water pools as a function of the pH_st with which the aqueous micelles are prepared. The significance of such data for interpreting the behaviour of enzymes confined in the micelles water pool is discussed. Based on the pH_wp scale, the apparent pK_a of phenol-red and 4-nitrophenol were determined in reverse micelles containing different buffers and different water content. The pK_a values obtained were rather sensitive to changes of both these factors, which was taken to signify that organic dies have only a very limited applicability to measure the acidity of the water pools of reverse micelles.     

Synthesis of a common trisaccharide fragment of glycoforms of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide

The first synthesis of the common trisaccharide of glycoforms of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide is reported. A fully protected trisaccharide precursor was prepared via a highly efficient α-(1→4)-glucosylation of a β-(1→3)-linked 6-O-benzyl-2-azido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-α-d-galactopyranoside. In contrast, an alternative sequence of glycosylations, which involves β-glucosylation of an α-(1→4)-linked Glc-GalN₃ unit, did not lead to the target trisaccharide backbone. Further O-deacetylation, azido group reduction and debenzylation of the protected trisaccharide precursor gave the corresponding trisaccharide amine. The latter structure was used in the synthesis of a series of trisaccharides bearing an acetyl group, an l-alanine or an N-acetylated l-alanine residue on its amino group at C-2 of GalN.     

Microscopic spacial effect on the dispersion polymerization in scCO2

We report for the first time the microscopic spacial effect on the dispersion polymerization of methyl methacrylate (MMA) in supercritical carbon dioxide (scCO₂). A variety of different-sized high-pressure vessels including microstructured holey optical fiber were employed to conduct the polymerization reactions. The molecular weights of the polymer products indicate that the function of the stabilizer and the process of chain growth are not significantly influenced. However, the SEM images show a gradual loss of the controlled morphology for the polymer products in reactors of dimension less than 1 mm under the same reaction conditions. This study provides a better understanding of the mechanism of the dispersion polymerization progress and gives a very important caution on the performance of microreactors.