Prefractionation techniques in proteome analysis: the mining tools of the third millennium

The present review deals with prefractionation protocols used in proteomic investigation in preparation for mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) map analysis. Briefly, reported methods focus on cell organelle differential centrifugation and on chromatographic approaches, to continue in extenso with a panoply of electrophoretic methods. In the case of chromatography, procedures useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins, revealed several hundreds of new species, previously undetected in unfractionated samples. Novel chromatographic prefractionation methods are also discussed such as a multistaged fractionation column, consisting in a set of immobilized chemistries, serially connected in a stack format (an assembly of seven blocks), each capable of harvesting a given protein population. Such a method significantly simplifies the complexity of treated samples while concentrating species, all resulting in a larger number of visible proteins by MS or 2-DE. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separation are multichamber apparatus, such as the multicompartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads. Multicup device electrophoresis and several others, exploiting the conventional technique of carrier ampholyte focusing, are reviewed. This review also reports approaches for sample treatments in order to detect low-abundance species. Among others, a special emphasis is made on the reduction of concentration difference between proteins constituting a sample. This latter consists in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of 20 amino acids, resulting in millions of different structures. When these beads are impregnated with complex proteomes (e.g., human sera) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility to evidence low-abundance species. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".     

A new cobalt carbonyl-carbon disulphide cluster containing a Co3C and a Co3S pyramid symmetrically linked by a CS2-bridge: sym-SCo6(CO)16C(CS2)

sym-(CO)₉Co₃C(CS₂)Co₃(CO)₇S has been isolated from the mixture formed in the reaction of Co₂(CO)₈ with CS₂. This is the fifth member of the C_mS_nCo_p(CO)_q family identified by X-ray diffraction studies. The molecular structure of the title compound is discussed and briefly compared with that of the asymmetric isomer and related compounds.     

Separation of charged and neutral isotopic molecules by micellar electrokinetic chromatography in coated capillaries

Some unique separations are reported of pairs of deuterated and non-deuterated compounds by capillary zone electrophoresis (CZE) in coated capillaries in the absence and presence of surfactant micelles. Pyridine (pyridine-h₅) and [² H₅]pyridine (pyridine-d₅) could be separated in plain buffer (R = 1.1) and in 2% Nonidet P-40 (R = 1.5). Owing to the good separation obtained, it was possible to assess the degree of cross-contamination when “pure” isotopes were analysed. A 1:1 mixture of benzoic-h₅ and -d₅ acid was poorly separated in the absence of detergent (R = 0.39) but well separated in 50 mM sodium dodecyl sulphate (SDS) (R = 1.14). Benzyl-h₅ and -d₅ alcohol were reasonably well separated in a micellar system containing 50 mM SDS (R = 1.02) (a separation previously unreported in the literature. Benzene-h₆ and -d₆ were well separated again in presence of 50 mM SDS (R = 1.59). It appears that CZE offers a unique environment for the separation of positively and negatively charged and neutral isotope mixtures, previously reported only using RP-HPLC and GC.     

A kinetic study of S-nitrosothiol decomposition

Under anaerobic conditions S-nitrosothiols 1a-e undergo thermal decomposition by homolytic cleavage of the S-N bond; the reaction leads to nitric oxide and sulfanyl radicals formed in a reversible manner. The rate constants, k(t), have been determined at different temperatures from kinetic measurements performed in refluxing alkane solvents. The tertiary nitrosothiols 1c (k1(69 degrees C) = 13 x 10(-3) min(-1)) and 1d (k1(69 degrees C) = 91 x 10(-3) min(-1)) decomposed faster than the primary nitrosothiols 1a (k1(69 degrees C) = 3.0 x 10(-3) min(-1)) and 1b (k1(69 degrees C) = 6.5 x 10(-3) min(-1)). The activation energies (E# = 20.5-22.8 Kcal mol(-1)) have been calculated from the Arrhenius equation. Under aerobic conditions the decay of S-nitrosothiols 1a-e takes place by an autocatalytic chain-decomposition process catalyzed by N2O3. The latter is formed by reaction of dioxygen with endogenous and/or exogenous nitric oxide. The autocatalytic decomposition is strongly inhibited by removing the endogenous nitric oxide or by the presence of antioxidants, such as p-cresol, beta-styrene, and BHT. The rate of the chain reaction is independent of the RSNO concentration and decreases with increasing bulkiness of the alkyl group; this shows that steric effects are crucial in the propagation step.     

Development of an integrated capillary electrophoresis/sensor for L-ascorbic acid detection

A CE/biosensor for measuring ascorbic acid was developed by coupling a polyaniline optical sensor and capillary electrophoresis (CE). The capillary column was partially modified with a thin film of polyaniline redox sensitive material. Ascorbic acid was detected by monitoring the changes in optical absorbance occurring to the polyaniline film upon the reduction reaction. The sensor response (change in optical absorbance at 650 nm) is proportional to the concentration of ascorbic acid over a range of 2.5-250 mg/L and the response range has shown a clear dependence on the characteristics of the polymerized film. High specificity and sensitivity of the present method, low sample consumption, short times of response (ca. 2 min) and the reproducibility of the results demonstrate that the CE/polyaniline-sensor could be further employed in the study of the relation between the content of L-ascorbic acid in body fluids and clinical parameters, e.g., cell ageing.     

Radical Sequential Processes Promoted by 1,5-Radical Translocation Reaction: Formation and [3 + 2] Anulation of Alkenesulfanyl Radicals

Radical addition of 2-substituted ethanethiols 1-5 to alkyl-, dialkyl-, and phenylacetylenes affords the corresponding beta-sulfanylalkenyl radicals, which can undergo 1,5-radical translocation (RT reaction) in competition with intermolecular hydrogen abstraction (HA reaction). The RT reaction is the first step of a sequential radical process leading to alkenesulfanyl radicals through an "intermolecular sulfanyl radical transaddition" from an alkene to an alkyne molecule. Alkenesulfanyl radicals can undergo a regioselective [3 + 2] anulation reaction with a CC triple bond, eventually leading to thiophene products through 5-endo cyclization of vinyl radicals onto CC double bond. The effect of the nature of ethanethiol and alkyne substituents on the RT/HA ratio has been investigated, and results will be discussed.     

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. IV: pH 6-8 intervals

Four commercial brands of carrier ampholytes (Ampholine, Pharmalyte, Servalyt, Bio-Lyte), in the pH 6-8 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by CE-MS of 10 out of 20 fractions harvested, in the second dimension. The findings: Ampholine (pH 6-8) contains 80 different M(r) compounds, in the M(r) interval 216-979 Da, for a total of 326 isoforms. Bio-Lyte (pH 6-8) consists of 62 different M(r) species, in the M(r) range 341-1048 Da, for a total of 237 isoforms. Servalyt (pH 6-8) is made of 126 different M(r) compounds, in the M(r) interval 240-785 Da, for a total of 703 isoforms. Pharmalyte (pH 5-8) comprises 123 amphoteres, in the M(r) range of 221-992 Da, for a total of 476 isoforms. Pharmalyte appears to be the best brand, with a good proportion of species focusing sharply at their pI position and relatively few 'poor' species, distributed along the entire pH gradient. General conclusions are drawn on the properties of all the pH intervals explored in this series of investigations and some guidelines for possible synthetic routes ameliorating the neutral and alkaline pH intervals are discussed.     

Volatiles emission patterns of different plant organs and pollen of Citrus limon

The volatiles emitted in vivo by different plant parts of Citrus limon (Rutaceae) have been identified by mean of head space-solid phase micro extraction (HS-SPME) and gas chromatography coupled with mass spectrometry (GC-MS) analyses. In particular, the profiles of flower buds, mature flowers, petals, stamens, gynaecium, pericarp of unripe and ripe fruits, young and adult leaves and pollen have been examined. Furthermore, the essential oil obtained from expression of ripe pericarp was studied. Volatiles were produced in distinctive amounts by the different plant organs, creating an interesting contrast, particularly within the flower parts: the highest amount of limonene (62.5%) was emitted by gynaecium, followed by stamens (22.9%) and petals (3.1%). Pollen did not produce limonene at all. The same compound is contained in higher amounts in the young leaves than in old ones (65.3% versus 30.1%). A possible defensive role of limonene and other volatiles, mainly terpene aldehydes, produced by young leaves has been hypothesized.     

The vibrational spectra of the cyanide ligand revisited. Bridging cyanides

The nu(CN) vibrational spectra of cyanide groups bridging two metal atoms present a confused picture. Factors relevant to the interpretation of the available data are reviewed. Some mechanisms for frequency change, relative to the corresponding terminal species, are made more quantitative than previously described, and others are highlighted for the first time. The kinematic effect is much less important than previously assumed. It seems that an effect responsible for the major part of the frequency increase upon bridging commonly observed is the cation electric field, together with concomitant relaxation (the IVSE model). However, a contribution may well come from the change in the sigma bonding in the CN unit.