Analysis of high molecular mass proteins larger than 150 kDa using cyanogen bromide cleavage and conventional 2-DE

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.     

Observation and calculation of the Cs2 2 3Delta(1g) and b 3Pi(0u) states

The Cs(2) 2 (3)Delta(1g) and b (3)Pi(0u) states have been observed by infrared-infrared double resonance spectroscopy for the first time. 221 2 (3)Delta(1g)<--A (1)Sigma(u) (+)<--X (1)Sigma(g) (+) double resonance lines have been assigned to transitions into the 2 (3)Delta(1g) v=6-13 vibrational levels. Resolved fluorescence into the b (3)Pi(0u) v(')=0-48 levels has been recorded. Molecular constants and potential energy curves are determined by the global fit of the entire set of the experimental data. Theoretical potential energy curves of the 2 (3)Delta(g) and b (3)Pi(u) states have been determined in the framework of the pseudopotential method and are compared with the experimental results.     

Thermodynamics of hydrophobic interaction chromatography of acetyl amino acid methyl esters

Thermodynamic analysis of hydrophobic interaction chromatography of amino acid methyl esters showed entropy-driven adsorption, consistent with solvophobic theory, except for phenyl ester on the Toyopearl resins. All esters adsorbed more strongly to the Toyopearl resins, including the polymethacrylate base matrix, than to Butyl Sepharose. Enthalpy changes were more favorable with the former, explaining the retention difference between Toyopearl Butyl and Butyl Sepharose. An enthalpy change versus heat capacity change plot showed Van der Waals interactions predominantly with the resin matrix. Literature data revealed the same effect for dansylamino acids, shown by isothermodynamic temperature analysis to adsorb more entropically than the esters.     

Structure, microstructure, and size dependent catalytic properties of nanostructured ruthenium dioxide

Nanostructured powders of ruthenium dioxide RuO₂ were synthesized via a sol gel route involving acidic solutions with pH varying between 0.4 and 4.5. The RuO₂ nanopowders were characterized by X-ray diffraction, scanning and transmission electron microscopy (SEM and TEM). Rietveld refinement of mean crystal structure was performed on RuO₂ nanopowders and crystallized standard RuO₂ sample. Crystallite sizes measured from X-ray diffraction profiles and TEM analysis varied in the range of 4-10 nm, with a minimum of crystallite dimension for pH=1.5. A good agreement between crystallite sizes calculated from Williamson Hall approach of X-ray data and from direct TEM observations was obtained. The tetragonal crystal cell parameter (a) and cell volumes of nanostructured samples were characterized by values greater than the values of standard RuO₂ sample. In addition, the [Ru-O₆] oxygen octahedrons of rutile structure also depended on crystal size. Catalytic conversion of methane by these RuO₂ nanostructured catalysts was studied as a function of pH, catalytic interaction time, air methane composition, and catalysis temperature, by the way of Fourier transform infrared (FTIR) spectroscopy coupled to homemade catalytic cell. The catalytic efficiency defined as FTIR absorption band intensities I(CO₂) was maximum for sample prepared at pH=1.5, and mainly correlated to crystallite dimensions. No significant catalytic effect was observed from sintered RuO₂ samples.     

UV-A induced oxidative stress is more prominent in naturally pigmented aged human RPE cells compared to non-pigmented human RPE cells independent of zinc treatment

To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.     

Influence of mass resolution on species matching in accurate mass and retention time (AMT) tag proteomics experiments

Diverse mass spectrometric instruments have been used to provide data for accurate mass and retention time (AMT) tag proteomics analyses, including ion trap, quadrupole time-of-flight, and Fourier transform mass spectrometry (FTMS). An important attribute of these instruments, beside mass accuracy, is their spectral resolution. In fact, the ability to separate peaks with close m/z values is likely to play a major role in enabling species identification and matching in analyses of very complex proteomics samples. In FTMS, resolution is directly proportional to the detection period and can therefore be easily tuned. We took advantage of this feature to investigate the effect of resolution on species identification and matching in an AMT tag experiment. Using an Arabidopsis thaliana chloroplast protein extract as prototypical 'real-life' sample, we have compared the number of detected features, the optimal mass tolerance for species matching, the number of matched species and the false discovery rate obtained at various resolution settings. It appears that while the total number of matches is not significantly affected by a reduction of resolution in the range investigated, the confidence level of identifications significantly drops as evidenced by the estimated false discovery rate.     

Isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of Burkholderia thailandensis E264

Lasso peptides are a structurally unique class of bioactive peptides characterized by a knotted arrangement, where the C-terminus threads through an N-terminal macrolactam ring. Although ribosomally synthesized, only the gene cluster for the best studied lasso peptide MccJ25 from Escherichia coli consisting of the precursor protein McjA and the processing and immunity proteins McjB, McjC, and McjD is known. Through genome mining studies, we have identified homologues of all four proteins in Burkholderia thailandensis E264 and predicted this strain to produce a lasso peptide. Here we report the successful isolation of the predicted peptide, named capistruin. Upon optimization of the fermentation conditions, mass spectrometric and NMR structural studies proved capistruin to adopt a novel lasso fold. Heterologous production of the lasso peptide in Escherichia coli showed that the identified genes are sufficient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related Burkholderia and Pseudomonas strains. In general, our rational approach should be widely applicable for the isolation of new lasso peptides to explore their high structural stability and diverse biological activity.     

β-N-Heterocyclic Cyclobutane Carboximides: Synthesis through a Tandem Base-Catalyzed Amidation/aza-Michael Addition Protocol and Facile Transformations

A stereoselective one-pot double derivatization of cyclobutene-1-carboxylic acid via a mild organic base catalyzed amidation/aza-Michael addition of benzo[d]oxazol-2(3H)-ones has been developed. This unprecedented tandem reaction provides access to novel β-N-heterocyclic cyclobutane carboximide derivatives with a trans geometry. The carboximide moiety reacts smoothly with nucleophiles, allowing access to diverse derivatives of trans-β-N-heterocyclic cyclobutanecarboxylic acid, including peptidomimetic structures.     

Microcins, gene-encoded antibacterial peptides from enterobacteria

Microcins are gene-encoded antibacterial peptides, with molecular masses below 10 kDa, produced by enterobacteria. They are secreted under conditions of nutrient depletion and exert potent antibacterial activity against closely related species. Typical gene clusters encoding the microcin precursor, the self-immunity factor, the secretion proteins and frequently the post-translational modification enzymes are located either on plasmids or on the chromosome. In contrast to most of the antibiotics of microbial origin, which are non-ribosomally synthesized by multimodular enzymes termed peptide synthetases, microcins are ribosomally synthesized as precursors, which are further modified enzymatically. They form a restricted class of potent antibacterial peptides. Fourteen microcins have been reported so far, among which only seven have been isolated and characterized. Despite the low number of known representatives, microcins exhibit a diversity of structures and antibacterial mechanisms. This review provides an updated overview of microcin structures, antibacterial activities, genetic systems and biosyntheses, as well as of their mechanisms of action.