Hansenmathiassen1058

The hydroperoxo iron(III) intermediate P450cam Fe(III) -OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450cam , could be transiently observed in the peroxo-shunt oxidation of the substrate-free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpd 0 enabled us to kinetically examine the formation and reactivity of P450cam Fe(III) -OOH species as a function of varying reaction conditions, such as pH, and concentration of H2 O2 , camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate-free form of P450cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid-base catalyst and is mainly governed by the ability of H2 O2 to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p$K\rm P450\hfill \atop \rm a\hfill$. Notably, no spectroscopic evidence for the formation of either Cpd I or Cpd II as products of heterolytic or homolytic OO bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1R)-camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe(III) -OOH intermediate in the oxidation of the natural substrate of P450cam .Mediastinal parathyroid cysts (PC) are rare, benign lesions, reported in fewer than 150 cases worldwide. Although most are asymptomatic and discovered incidentally on imaging, symptoms of dyspnea, dysphagia, hoarseness, palpitations, hypercalcemia, and innominate or jugular venous thrombosis have been reported. Sternotomy or thoracotomy has traditionally been the approach used to resect mediastinal PCs. We describe the first reported case of a robot-assisted resection of a mediastinal PC.Grain refinement via semi-solid deformation is desired to obtain superior mechanical properties of cast components. Using quantitative in situ synchrotron X-ray tomographic microscopy, we show an additional mechanism for the reduction of grain size, via liquation assisted transgranular cracking of semi-solid globular microstructures. Here we perform localized indentation of Al-15wt.%Cu globular microstructures, with an average grain size of ∼480 μm, at 555 °C (74% solid fraction). Although transgranular fracture has been observed in brittle materials, our results show transgranular fracture can also occur in metallic alloys in semi-solid state. This transgranular liquation cracking (TLC) occurs at very low contact stresses (between 1.1 and 38 MPa). With increasing strain, TLC continues to refine the size of the microstructure until the grain distribution reaches log-normal packing. The results demonstrate that this refinement, previously attributed to fragmentation of secondary arms by melt-shearing, is also controlled by an additional TLC mechanism.It has been previously reported that the glycosylation site and protein-sequence information could be obtained for ribonuclease B by top-down electron-capture dissociation (ECD) and collision-induced dissociation (CID) mass spectrometry (MS). However, the sequence coverage of ribonuclease B was limited in a single activation, and the structural information on the glycan moiety was not probed successfully in previous experiments. Here, we demonstrate that ECD and CID techniques can be used together as an effective top- down method for the structural characterization of intact glycoprotein. Even without an elaborate pre- or post- ECD activation, a high sequence coverage ( less then 90%) of ribonuclease B could be achieved with substantial amounts of structural information for the glycan moiety. By comparing our work with previous results, it is postulated that the disulfide bond reduction strategy might play a significant role in determining the efficiency of top-down MS.Cyclodextrins (CDs) are a group of nontoxic oligosaccharides that are widely used as drug excipients and protein stabilizers. CDs have also been found to reduce the neurotoxicity and fibrillation of amyloid beta (Aβ), the major component of the amyloid plaques found in the brain of patients suffering from Alzheimer's disease. The formation of these plaques was found to be enhanced by the presence of iso-aspartic acid (isoAsp) residues in the Aβ peptide, which can be formed by deamidation from asparagine (Asn). To explore further the influence of CDs on Aβ, we investigated three Asn-containing peptides, including Aβ25-35, by electrospray ionization, electron capture dissociation, and Fourier-transform ion cyclotron resonance mass spectrometry to explore details of the deamidation process in the presence and absence of peptide/CD adducts. The results showed that CDs reduced the formation of the isomerization product isoAsp during peptide deamidation. This finding might help to better understand the role of CDs during the protein-aggregation process.The chemical analysis of tartaric acid (TA) and syringic acid (SA), as grape product markers in ancient ceramic vessels from the sites of Manduria and Torre di Satriano (southern Italy), was successfully performed. Firstly, the fragmentation behaviour of TA and SA as deprotonated molecules, [M-H](-), obtained by collision-induced dissociation, was investigated. Then, reversed-phase liquid chromatography (RPLC) with electrospray ionization (ESI) in negative ion mode, using a quadrupole linear ion trap in multiple reaction monitoring (MRM), was employed. A binary mobile phase composed of water-acetonitrile with 0.1% (v/v) acetic acid enabled the optimum ESI efficiency of SA, greatly improving its identification when it occurs in trace amounts. Chemical analysis of ancient pottery fragments is a valid method for establishing the existence of preserved organic residues, which is valuable new evidence for the culture and customs of ancient populations, in this case those of southern Italy. The proposed RPLC-ESI-MRM method allowed a systematic investigation of ceramic fragments of both archaeological sites, thus providing positive evidence for the presence of TA and SA as grape product markers in storage vessels dating back to the ninth to third centuries BC.Proteomic approach in combination with mass spectrometry demonstrates a great potential for identification of proteinaceous materials in works of art. In this study we used a linear trap quadrupole Orbitrap (LTQ-Orbitrap), a state-of-the-art mass spectrometer for parts per million accuracy analyses of peptides behind tryptic hydrolysis. After the efficiency of the proteomic method was confirmed for reference and model samples, micro-samples from historical paintings were for the first time analysed using this technique. Superior performances of the liquid chromatography-mass spectrometry approach using a LTQ-Orbitrap mass spectrometer allowed identification of egg yolk peptides in two samples from nineteenth-century Orthodox icons, indicating egg tempera as the painting technique. Accurate precursor ion masses, in the range of ±2 ppm, and retention times of tryptic peptides strengthen protein identification. Additionally, in all historical samples the presence of animal glues suggested that the ground layer was likely bound using bovine collagen. Comparing to results acquired using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry in our previous study, here we achieved higher ion scores and protein scores, better sequence coverage and more identified proteins. In fact, a combination of the two mass spectrometric techniques provided overlapping and complementary data, related to the detection of peptides with different physicochemical properties.This study examines the non-covalent interactions between glycosaminoglycan (GAG) oligosaccharides using nanoelectrospray ionization mass spectrometry (nanoESI-MS). It is the first time that interactions between oligosaccharides have been observed using MS. The importance of interactions between GAGs has recently attracted much interest because they are related to biological functions. For instance, hyaluronic acid (HA) is known to associate with chondroitin sulfates (CSs), although the details of the interaction remain unclear. In general, non-covalent interactions between glycans are too weak to detect by general means. In this work, we applied nanoESI-MS with high sensitivity, which is widely used to observe non-covalent interactions, to investigate the interaction between HA and CSs. HA and CS oligosaccharides are used to discuss the results in a simplified manner. Our approach is aimed at interpreting the behavior of GAG polysaccharides from the information obtained using the oligosaccharides. HA and CS tetrasaccharides were demonstrated to associate to form heterodimer ions that were easily detected using nanoESI-MS. We also determined the stoichiometry of the interaction and calculated the K(d) values of the interactions between HA and CS tetrasaccharides. How these structures affect the strength and stability of the non-covalent complexes is discussed. Further study of the interactions between HA and CS oligosaccharides will clarify the biological meaning of the coexistence of HA and CS in body fluids and tissues.5-Methylcytosine (5-MC) is an important epigenetic modification of DNA. Abnormally high concentrations of this substance appear because of the hypermethylation of cytosine. Therefore, the measurement of the quantity of this compound in mammals is of great importance. Recently, we reported that several imidazolium-based zwitterionic sulfonates form complexes with 5-MC in solution, which can be studied by electrospray ionisation mass spectrometry (ESI-MS). It is shown in this paper that such an association can be utilised for the detection of 5-MC in a DNA sample using high-throughput a flow injection analysis ESI-MS method. A variety of the sulfonate zwitterions have been tested as m/z shift reagents to increase the selectivity of the analysis. It is shown that either of the zwitterions can be used without the loss of sensitivity. JAK inhibitor The performance of the method was tested in terms of linearity range, sensitivity, intra- and between-day precision and accuracy, matrix effect and carryover. The method described is characterised by simplicity, a good limit of quantitation (1 pg injected) and low run times (at least 50 injections per hour). In addition, high-performance liquid chromatography and tandem mass spectrometry are not required. The possibility exists to widen the scope of the method to other amidine-containing compounds present in more complicated matrices.The gas-phase dissociation pathways of proteins/peptides are usually affected by the nature of the charge carrier and the sequence of amino acid residues. The effects of peptide structural parameters, including peptide composition, chain length and amide hydrogen, on the gas-phase dissociation of Cu(II)-model peptide complexes were explored in this study. Polyglycine peptides with flexible frames were used as probes to reduce the complexity of the system and illustrate the mechanism. Results revealed that the types of fragment ions generated in the electron capture dissociation (ECD) of Cu(II)-adducted peptides changed according to the basic amino acid residue composition. Charged or neutral tryptophan side-chain losses were observed in the collision-induced dissociation (CID) of Cu(II)-peptide complexes. Internal electron transfer between tryptophan and metal ion within the complex occurred during the CID reaction, leaving the charge-reduced Cu(+) as a closed d-shell stable electron configuration. The choice of the reaction channel was then determined by the gas-phase basicity of the peptide.