A multiplexed method for performing MS/MS on multiple ions simultaneously in a miniature rectilinear ion trap (RIT) mass spectrometer has been developed. This method uses an ion encoding procedure that relies on the mass bias that exists when ions are externally injected into an RIT operated with only a single phase RF applied to one pair of electrodes. The ion injection profile under such conditions ions is Gaussian-like over a wide range of RF amplitudes, or low mass cutoff (LMCO) values, during ion accumulation. We show that this distribution is related to ion m/z and is likely caused by ions having an optimal range of pseudo-potential well depths for efficient trapping. Based on this observation, precursor ion intensity changes between two different injection LMCO values can be predicted, and these ion intensity changes are found to be carried through to their corresponding product ions, enabling multiplexed MS/MS spectra to be deconvoluted. The gas-phase reactions of a series of coordinatively unsaturated [Ni(L)n]y+ complexes, where L is a nitrogen-containing ligand, with chemical warfare agent (CWA) simulants in a miniature rectilinear ion trap mass spectrometer were investigated as part of a new approach to detect CWA. Results show that the metal complex ions can react with low concentrations of several CWA simulants, including dipropyl sulfide (simulant for mustard gas), acetonitrile (simulant for the nerve agent tabun), and diethyl phosphite (simulant for nerve agents sarin, soman, tabun, and VX), thereby providing a sensitive means of detecting these compounds. The [Ni(L)n]2+ complexes are found to be particularly reactive with the simulants of mustard gas and tabun, allowing their detection at low parts-per-billion (ppb) levels. These detection limits are well below the median lethal doses for these CWAs, which indicates the applicability of this new approach, and are about two orders of magnitude lower than electron ionization detection limits on the same mass spectrometer. The use of coordinatively unsaturated metal complexes as reagent ions offers the possibility of further tuning the ion-molecule chemistry so that desired compounds can be detected selectively or at even lower concentrations. Mass spectrometry has become a tool for studying noncovalently bound complexes. Specifically, electrospray ionization mass spectrometry (ESI-MS) has found increasing use for the determination of affinity (Ka) or dissociation (Kd) constants. Direct measurement of the equilibrium components by ESI-MS is the most straightforward approach for determining binding equilibrium constants, but this approach is prone to error and has some inherent limitations. Transferring complexes from solution to the gas phase may perturb the equilibrium concentrations and/or different ionization efficiencies may cause the resulting ion signals not to reflect actual solution concentrations. Furthermore, ESI only works under a limited range of solvent conditions (i.e. low ionic strengths), which limits the broad applicability of this approach. We propose an approach based on covalent labeling in the context of metal-catalyzed oxidation (MCO) reactions that, when combined with MS, overcomes such limitations when determining metal-ligand binding constants. The MCO-MS approach will provide concurrent information regarding metal binding site and metal-protein binding affinity. Optimization of the MCO reaction through isotopic mass tags will permit enhanced identification of modified residues. Application of this method to study the affinity and binding interactions of other divalent metals with β2m are likely to provide insight into the specificity of copper for causing β2m amyloid formation.

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Metal contamination is a considerable environmental problem because metals are persistent contaminants. Ion exchange is one of the most commonly used treatment options for trace metal removal. This research develops and evaluates a redox active modified ion exchange system that has the potential to reduce the ionic strength of ion exchange regeneration streams. Poly-L-cysteine (PLC) was selected as the redox active, adsorbing functional group on the surface of a reticulated vitreous carbon (RVC) electrode. PLC is an excellent soft acid metal chelator and is unique in that its thiol groups can form disulfide bonds with each other. The reduction of available thiols changes the metal binding capacity of the peptide since the thiol is the primary binding group. RVC provides a macroporous conductive monolithic resin to support the peptide. An experimental apparatus was designed to study the properties of this system and estimate performance. Distinct oxidized and reduced states of PLC on the surface of the RVC were confirmed by changes in metal binding characteristics. Adsorption edges showed a sharper pH dependence for the reduced electrode compared to the oxidized electrode from pH 3-7. Adsorption isotherms performed at pH 7 showed increased capacity for the reduced electrode. The change was reversible by chemical and electrical reduction. This difference was confirmed at the molecular level with Cd- EXAFS of oxidized and reduced electrodes. A greater degree of cadmium-sulfur coordination was observed on the reduced electrode and a greater cadmium-oxygen coordination was apparant on an oxidized electrode. A multidentate adsorption model was developed to model the pH dependent behavior of cadmium adsorption on the PLC-RVC surface. Nickel adsorption showed increased adsorption in the oxidized state. The most likely explanation is increased carboxylate complexation. The electronically switchable ion exchange system (ESIE) provides a framework for modifying traditional ion exchange processes. The system has 5 to 10 times less specifc capacity than current ion exchange systems, but uses solutions 10-100 times lower in ionic strength for regeneration. Further studies on the effect of ionic strength on adsorption and current usage are necessary to compare the cost of the ESIE process to traditional ion exchange. / text

CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across cellular membranes. CopA contains at least two metal binding domains, a regulatory N-terminal Metal Binding Domain (N-MBD) and an occlusion/coordinating metal binding site in the 6th, 7th and 8th transmembrane segments. Previous studies showed that the presence of millimolar concentration of Cys is essential for CopA activity. The high affinity of CopA for metal in the presence of millimolar concentration of Cys suggests a multifaceted interaction of the enzyme with Cys. To elucidate the role of Cys, we studied its effect on the partial reactions of the catalytic cycle of CopA. We observed that 2-50 mM Cys accelerates enzyme turnover with little effect on the Cu+ affinity of CopA. Cys accelerates enzyme phosphorylation, but has no effect on the dephosphorylation rates. Thus, Cys increases steady state phosphoenzyme levels. Besides, Cys has no significant effect on E1¡ÃƒÂªE2 equilibrium. Similar results were observed in truncated CopA lacking the N-MBD suggesting that enzyme activation by Cys is independent of the regulatory metal binding sites. These results and the kinetic analysis of activation curves suggest that while Cu+ is delivered to the transport site as a Cu-Cys complex, Cys in the mM range stimulates the ATPase acting as a non-essential activator.

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Dans cette étude, le transporteur ABC de zinc de Streptococcus agalactiae, première cause d’infections materno-foetale en France, a été caractérisé. Nous avons montré que ce transporteur se compose, du complexe perméase-ATPase AdcCB, associé à trois protéines membranaires Lmb, AdcA et AdcAII redondantes dans la fixation de zinc. Ce transporteur comporte également deux protéines Sht et ShtII, retrouvées au niveau de la paroi, et nécessaires aux protéines Lmb et AdcAII pour la capture de zinc. L’absence d’un transporteur fonctionnel, par la triple délétion des gènes lmb, adcA et adcAII ou du complexe adcCB, a révélé une inhibition de la croissance et une perturbation de la division de la bactérie lorsqu’elle se trouve dans un environnement carencé en zinc. De plus, nous avons montré que ce transporteur de zinc participe à la survie de la bactérie en milieux biologiques humains, comme le liquide amniotique ou le LCR, où la bactérie est retrouvée lors d’infections, suggérant l’importance du transporteur lors du processus infectieux. Ces résultats ont mis en évidence, pour la première fois, que le zinc assure des fonctions biologiques vitales pour S. agalactiae et que, dans des conditions de forte carence en zinc, le transporteur Adc/Lmb représente le principal système d’acquisition de zinc de la bactérie. / In this study, the zinc-ABC transporter of Streptococcus agalactiae, the first cause of materno-foetal infections in France, was characterized. We showed that this transporter is composed of an AdcCB permease-ATPase complex in association with three membrane-associated proteins Lmb, AdcA and AdcAII, which are redundant in zinc-binding. This transporter also possesses two proteins Sht and ShtII, which are associated to the cell wall, and that are necessary for the Lmb and AdcAII proteins for zinc capture. The absence of a functional transporter, by the triple deletion of the lmb, adcA and adcAII genes or the adcCB complex, revealed a growth inhibition and a disruption of the division of the bacterium when it is in a zinc-restricted environment. Furthermore, we showed that the zinc-ABC transporter contributes to the survival of the bacterium in human biological fluids, as the amniotic fluid or the cerebrospinal fluid, where the bacterium is found during infections, suggesting the importance of the transporter during the infectious process. These results hightlighted, for the first time, that zinc has biologically vital functions in S. agalactiae and that, under high zinc deficiency conditions, the Adc/Lmb transporter is the main zinc acquisition system of the bacterium.

Ce travail porte sur la synthèse chimique d’analogues simples d’anthocyanes, une classe majeure de pigments naturels solubles dans l’eau. Onze ions flavylium substitués par des groupements hydroxyl,méthoxyl et beta-D-glucopyranosyloxyl en positions 4’, 5 et 7 ont été préparés en utilisant des procédures simples. De plus, les deux principales 3-désoxyanthocyanidines du sorgho rouge, l’apigéninidine (APN) et la lutéolinidine (LTN), ont été synthétisées en une seule étape. Les propriétés physico-chimiques ainsi que l’activité antioxydante ont été étudiées pour le chlorure de 3’,4’,7-trihydroxyflavylium (P1), son 7-O-beta-D-glucoside (P2) et le chlorure de 3’,4’,5,7-tétrahydroxyflavylium (LTN). Grâce à leur noyau catéchol, ces pigments complexent rapidement FeIII, AlIII and CuI et ne se lient que faiblement à FeII tout en stimulant son autoxydation en FeIII. Suite à la complexation de CuII, les pigments subissent une oxydation. Les aglycones P1 et LTN sont des ligands modérés de l’albumine de sérum humain (HSA) et leurs chalcones ont montré une plus grande affinité pour la HSA que leurs formes colorées. Leur capacité antioxydante a été démontrée par le test de réduction du radical stable DPPH et par l’inhibition de la peroxydation lipidique induite par le fer héminique, un modèle de stress oxydant postprandial dans l’estomac. Les aglycones P1 et LTN (particulièrement, dans leur forme incolore chalcone) sont plus efficaces que le glucoside P2. / This work deals with the chemical synthesis of simple analogs of anthocyanins, the main class of watersolublenatural pigments. Eleven flavylium ions with hydroxyl, methoxyl and beta-D-glucopyranosyloxylsubstituents at positions 4’, 5 and 7 have been prepared by straightforward chemical procedures.Moreover, the two main 3-deoxyanthocyanidins of red sorghum, apigeninidin (APN) and luteolinidin(LTN), have been synthesized in a one-step protocol. The physicochemical properties and antioxidantactivity are investigated for 3’,4’,7-trihydroxyflavylium chloride (P1), its 7-O-beta-D-glucoside (P2) and3’,4’,5,7-tetrahydroxyflavylium chloride (LTN). Owing to their catechol B-ring, they rapidly bind FeIII,AlIII and CuI, more weakly interact with FeII while promoting its autoxidation to FeIII. Following CuIIbinding, the pigments undergo oxidation. Aglycones P1 and LTN are moderate ligands of human serumalbumin (HSA) with chalcones having a higher affinity for HSA than the corresponding colored forms.The antioxidant activity of P1, P2 and LTN is investigated via two tests: reduction of the stable DPPHradical and inhibition of heme-induced lipid peroxidation (a model of postprandial oxidative stress inthe stomach). Aglycones P1 and LTN (especially in their colorless chalcone form) are more potent thanglucoside P2.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.