Search

International audience; Purpose: To date, the analysis of gadolinium (Gd) speciation in the brain of animals administered with macrocyclic and linear Gd-based contrast agents (GBCAs) has been limited to Gd soluble in mild buffers. Under such conditions, less than 30% of the brain tissue was solubilized and the extraction recoveries of GBCAs into the aqueous phase were poor, especially in the case of the linear GBCAs. The aim of this study was to find the conditions to solubilize the brain tissue (quasi-)completely while preserving the Gd species present. The subsequent analysis using size exclusion chromatography–inductively coupled plasma–mass spectrometry (SEC-ICP-MS) was intended to shed the light on the speciation of the additionally recovered Gd.Methods: Four groups of healthy female Sprague Dawley rats (SPF/OFA rats; Charles River, L'Arbresle, France) received randomly 5 intravenous injections (1 injection per week during 5 consecutive weeks) of either gadoterate meglumine, gadobenate dimeglumine, gadodiamide (cumulated dose of 12 mmol/kg), or no injection (control group). The animals were sacrifice 1 week (W1) after the last injection. Brain tissues were solubilized with urea solution, whereas tissues extracted with water served as controls. Total Gd concentrations were determined in the original brain tissue and its soluble and insoluble fractions by inductively coupled plasma–mass spectrometry (ICP-MS) to calculate the Gd accumulation and extraction efficiency. Size exclusion chromatography coupled to ICP-MS was used to monitor the speciation of Gd in the soluble fractions. The stability of GBCAs in the optimum conditions was monitored by spiking the brain samples from the untreated animals. The column recoveries were precisely determined in the purpose of the discrimination of weakly and strongly bound Gd complexes. The identity of the eluted species was explored by the evaluation of the molecular size and retention time matching with Gd chelates and ferritin standard. The speciation analyses were carried out for 2 different brain structures, cortex and cerebellum.Results: The combination of water and urea extractions (sequential extraction) managed to solubilize efficiently the brain tissue (97% ± 1%) while preserving the stability of the initially injected form of GBCA. For macrocyclic gadoterate, 97% ± 1% and 102% ± 3% of Gd initially present in the cortex and cerebellum were extracted to the soluble fraction. For gadobenate, similar amounts of Gd (49% ± 1% and 46% ± 4%) were recovered from cortex and cerebellum. For gadodiamide, 48% ± 2% of Gd was extracted from cortex and 34% ± 1% from cerebellum. These extraction efficiencies were higher than reported elsewhere. The SEC-ICP-MS and the column recovery determination proved that Gd present at low nmol/g levels in brain tissue was exclusively in the intact GBCA form in all the fractions of brain from the animals treated with gadoterate. For the linear GBCAs (gadobenate and gadodiamide), 3 Gd species of different hydrodynamic volumes were detected in the urea-soluble fraction: (1) larger than 660 kDa, (2) approximately 440 kDa, and (3) intact GBCAs. The species of 440 kDa corresponded, on the basis of the elution volume, to a Gd3+ complex with ferritin. Gd3+ was also demonstrated by SEC-ICP-MS to react with the ferritin standard in 100 mM ammonium acetate (pH 7.4). In contrast to macrocyclic gadoterate, for linear GBCAs, the column recovery was largely incomplete, suggesting the presence of free, hydrolyzed, or weakly bound Gd3+ with endogenous ligands.Conclusions: The sequential extraction of rat brain tissue with water and urea solution resulted in quasi-complete solubilization of the tissue and a considerable increase in the recoveries of Gd species in comparison with previous reports. The macrocyclic gadoterate was demonstrated to remain intact in the brain 1 week after administration to rats. The linear GBCAs gadobenate and gadodiamide underwent ligand exchange reactions resulting in the presence of a series of Gd3+ complexes of different strength with endogenous ligands. Ferritin was identified as 1 of the macromolecules reacting with Gd3+. For the linear GBCAs, 3% of the insoluble brain tissue was found to contain more than 50% of Gd in unidentified form(s).

International audience; Asphaltenes are considered to be the most problematic components of heavy oils because they can self-aggregate which leads to precipitation and causes various problems during oil recovery, transportation, and refining. The contribution of the porphyrins present in asphaltenes to the aggregation was previously studied by gel permeation chromatography inductively coupled plasma-mass spectrometry (GPC-ICP MS). The molecular weight of asphaltene aggregates was shown to be increased by free metal-containing porphyrins (corresponding to the lower molecular weight fraction) interacting with the aggregate’s surfaces by weak forces. The characterization of free porphyrins within the asphaltenes is therefore for the understanding of the mechanism of the aggregation, coprecipitation, and demetalation processes. Here, we developed a method for the separation of free porphyrins from asphaltenes on the basis of their polarity using high-performance thin-layer chromatography (HPTLC). This technique, using disposable plates, is particularly well suited for asphaltene analysis since it eliminates the risk of clogging typical of column chromatography. Cellulose plates were used in this study. The lower polarity of their hydroxyl groups limit the irreversible adsorption and improves the detection limit by the ICP-MS. Two well-separated peaks were obtained from purified asphaltene (Asphaltene 2017; Asphaltene Characterization Interlaboratory Study for PetroPhase 2017. In Proceedings of the 18th International Conference on Pretroleum Phase Behavior and Fouling, Le Havre, France, June 11–15, 2017; Total, the University of Pau, and the University of Rouen-Normandy: Le Havre, France, 2017.) and its corresponding whole crude oil and C5 and C7 fractions. The distribution of vanadium due to migration was determined by laser ablation (LA) ICP MS. The eluted fraction contained the free porphyrins, whereas the major fraction did not migrate and corresponded to trapped porphyrins. A comparison with the signal obtained by UV densitometry allowed the ratio between the inorganic and organic material to be measured.

The evolution of the field of element speciation, from the targeted analysis for specific element species toward a global exploratory analysis for the entirety of metal- or metalloid-related compounds present in a biological system (metallomics), requires instrumental techniques with increasing selectivity and sensitivity. The selectivity of hyphenated techniques, combining chromatography, and capillary electrophoresis with element-specific detection (usually inductively coupled plasma mass spectrometry, ICP MS), is often insufficient to discriminate all the species of a given element in a sample. The necessary degree of specificity can be attained by ultrahigh-resolution (R >100,000 in the m/z < 1,000 range for a 1 s scan) mass spectrometry based on the Fourier transformation of an image current of the ions moving in an Orbitrap or an ion cyclotron resonance (ICR) cell. The latest developments, allowing the separate detection of two ions differing by a mass of one electron (0.5 mDa) and the measurement of their masses with a sub-ppm accuracy, make it possible to produce comprehensive lists of the element species present in a biological sample. Moreover, the increasing capacities of multistage fragmentation often allow their de novo identification. This perspective paper critically discusses the potential state-of-the-art of implementation, and challenges in front of FT (Orbitrap and ICR) MS for a large-scale speciation analysis using, as example, the case of the metabolism of selenium by yeast.

Mycosporines and mycosporine-like amino acids have been described as natural sunscreens and antioxidant compounds presenting a great potential for health and cosmetic applications. Herein, an untargeted screening approach for mycosporines and mycosporine-like amino acids (MAAs) was developed by the coupling of zwitterionic hydrophilic interaction liquid chromatography (HILIC) with multistage electrospray mass spectrometry MS2/MS3 using an Orbitrap analyzer and fragment ion search (FISh). This method was applied to study the mycosporine and MAA contents of five algae extracted using a 50% methanol solution and sonication. Candidate-MAAs were detected by mining eight characteristic fragment ions in their HILIC data-dependent MS2 mass spectrum. Their exact masses were measured with 3 ppm mass accuracy and their structures were elucidated on the basis of the MS3/MS4 mass spectra. The method developed was validated with a targeted analysis using an extract of Gymnogongrus devoniensis which confirmed the detection of 14 MAAs reported in the literature. In addition, 23 previously unreported MAAs were detected and the structures could be assigned for seven of them. The developed method was applied to the analysis of four algae: Gelidium sesquipedale, Halopithys incurva, Porphyra rosengurtii and Cystoseira tamariscifolia allowing the detection of MAAs, including some reported here for the first time.

cited By 0; Article in Press; International audience; Medium and heavy crude oils and high-boiling distillation fractions which are increasingly used in petroleum industry contain high concentrations of sulfur and metals. Their behavior in refining processes is critically dependent on the speciation. Recent analytical developments, especially on the level of coupled techniques and high-resolution mass spectrometry, start allowing the speciation of individual metal compounds in crude oil known for its extreme complexity. These developments include: (i) GC stationary phases of high thermal stability and the high-temperature interfaces with ICP-MS and TOF-MS; (ii) high-efficiency microcolumn gel-permeation chromatography with detection by sector-field ICP-MS; (iii) thin layer chromatography coupling with laser ablation ICP-MS detection; and (iv) two-dimensional separation protocols increasing the purity of heteroelement containing fractions. Progress in electrospray and atmospheric-pressure photoionization Fourier Transform MS allows resolutions of above 1,000,000 to be achieved making it possible to identify by accurate mass measurement individual sulfur and metal species directly in crude oils. © 2017 Elsevier B.V.

BTEX (benzene, toluene, ethylbenzene, and the different xylene isomers), known for carcinogenic and neurotoxic effects, are common environmental contaminants. The first step for the development of the bioremediation technologies is the detection of intense microbial degradation in contaminated waters in the quest for the most active bacterial strains. This requires the multispecies analysis for BTEX metabolites which are considered as markers of microbial degradation. A direct (50 &micro, L injection) HPLC&ndash, electrospray MS/MS analytical method was developed for the simultaneous analysis of 11 BTEX metabolites (o-, m-, p-toluic, salicylic, benzoate, benzyl, and phenyl succinic acids, 2-(1-phenylethyl)-, 2-(2-methylbenzyl), and 2-(3-methylbenzyl)-, 2-(4-methyl benzyl)-succinic acids) in bacterial cultures and ground waters down to 0.1 ng/mL. The optimization of the chromatographic conditions allowed for the resolution of position isomers of toluic and methylbenzyl-succinic acids. The stability of the analytes during sample storage tested in different conditions showed the instability of some of them when stored at room temperature. The feasibility of the method was demonstrated by the detection of all the investigated metabolites in a water sample of a deep aquifer hosting natural gas storage. A model laboratory study emphasized the importance of 2-(2-methylbenzyl)-succinic acid as a marker of anaerobic microbial degradation.

International audience; A sequential fractionation procedure based on (i) water extraction, (ii) hexane extraction, (iii) saccharification, and (iv) proteolysis was developed to provide the first ever data on the molecular distribution of iron in maize. This was completed by the operational determination of the iron bioavailability using an in-vitro simulated model for gastro-intestinal digestion. The coupling of hydrophilic interaction chromatography (HILIC) and size exclusion chromatography (SEC) with the parallel detection by inductively coupled plasma mass spectrometry (ICP-MS) and high resolution electrospray mass spectrometry (HR-ESI-MS) allowed the identification of water-soluble Fe(III)-mugineate, Fe(III)-(citrate)2, and Fe(III)2-(phytate)2. The procedures were applied to study some well characterized maize varieties having shown previously differences in iron bioavailability during cell culture and animal model feeding studies. The combined analytical methods developed in this work could unambiguously discriminate low from high Fe bioavailable seeds in these closely related maize varieties.

International audience; Single particle inductively coupled plasma mass spectrometry (SP-ICP-MS) has become a well-established technique for the detection, size characterization and quantification of inorganic nanoparticles but its use for the analysis of micro- and nanoparticles composed of carbon has been scarce. Here, the analysis of a microplastic suspensions by ICP-MS operated in single particle mode using microsecond dwell times is comprehensively discussed. The detection of polystyrene microparticles down to 1.2 μm was achieved by monitoring the 13C isotope. Plastic microparticles of up to 5 μm were completely volatized and their components atomized, which allowed the detection of microplastics, their quantification using aqueous dissolved carbon standards, and the measurement of the size-distribution of the detected particles. Limits of detection of 100 particles per milliliter were achieved for an acquisition time of 5 min. The method developed was applied to the screening of microplastics in personal care products and released from food packagings. The chemical identity of the detected microplastics was confirmed by attenuated total reflectance Fourier-transform infrared spectroscopy.

OBJECTIVES: Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. Because the nature of this accumulated Se is not well characterized, we studied selenometabolomics in Se-deficient, Se-adequate, and high-Se liver. METHODS: Turkey poults were fed 0, 0.4, and 5 μg Se/g diet as Na2SeO3 in a Se-deficient (0.005 μg Se/g) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. RESULTS: In liver from turkeys fed a true Se-deficient diet and supplemented with inorganic Se (selenite), no selenomethionine (SeMet) was detected showing that the turkey cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient turkey liver, as expected in animals with negligible selenoprotein levels. Sec content in liver of turkeys fed high Se only doubled compared with Se-adequate liver, indicating that the 6-fold incryscease in liver Se in these birds was not due to increases in selenoproteins. What increased dramatically in high Se liver were the low molecular weight (MW) selenometabolites, glutathione-, cysteine- and methyl- conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). In addition, size-exclusion chromatography and followup analysis demonstrated that a substantial amount of Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide links, in addition to Sec-containing selenoproteins. In high-Se liver, these “selenosugar-decorated” proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to the low MW selenometabolites. CONCLUSIONS: In Se-adequate liver, far more Se is present as the selenosugar moiety, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols, leading to the formation of methyl-SeGalNac. This suggested pathway, underlying adaptation to high Se status in animals, needs further investigation including study of the potential of selenosugar compounds as biomarkers of high Se status. FUNDING SOURCES: USDA, Hatch project 1,004,389, and by the Wisconsin Alumni Foundation, Selenium Nutrition Research Fund 12,046,295.

International audience; An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS (78Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

International audience; Petroleum and its fractions are some of the most complex mixtures found in analytical chemistry. Mass spectrometry currently plays an increasing role in the characterization of these matrices. Since the last review on this topic in 2011, several new approaches have been introduced, and these approaches increasingly use sample fractionation by extraction and/or liquid chromatographic techniques. This review considers molecular mass spectrometry (with special emphasis on the use of ion mobility) and inorganic mass spectrometry. The combination of both techniques paves the way to "petrointeractomic" approaches, which are introduced as a novel, important part of "petroleomic" approaches.

cited By 0; Article in Press; International audience; Iron is an important nutrient essential for plants and critical for human health. The state-of-the art of methods for iron speciation in cereal grains and plant fluids is critically reviewed. Particular attention is given to the latest developments in the coupling of HPLC with the parallel ICP MS and electrospray ionization (ESI) MS/MS detection, usually QTOF MS or Q-Orbitrap MS, for the identification and quantification of iron species. The coupled techniques allow the direct microanalysis of plant intracellular fluids (xylem and phloem) and complement X-ray absorption spectroscopy (XANES and EXAFS). The increasing resolution and sensitivity of electrospray mass spectrometers and emergence of software allowing extraction of iron specific data from large chromatographic data sets are responsible for the growing role of electrospray MS/MS in speciation studies. The use of stable isotopes for the probing of the reactivity and stability of endogenous metal complexes and quantitative analysis are rising in importance.

cited By 0; Article in Press; International audience; Selenium is an essential element of which the deficiency is often compensated for by supplementation with products based on yeast grown on Se-rich media. Advances in HPLC with element- and molecule-specific detection by mass spectrometry have resulted in evidence of the presence of hundreds of selenocompounds in Se-rich yeast, with different properties and in a large concentration range. This spurs interest in extending -omics-type approaches to selenium speciation analysis to other samples with the ultimate goal to confer biological significance to the obtained data. This review critically evaluates the recent advances in selenium speciation analysis, especially in terms of sensitivity and specificity of ICP-MS and electrospray-MS, and, in terms of resolution, mass accuracy and multistage fragmentation by electrospray-MS. A focus is made on the evolution of the perception of the competitiveness and complementarity of ICP-MS and electrospray-MS detection in HPLC for qualitative and quantitative Se speciation analysis

Lanthanide polymer-labelled antibodies were investigated to improve the analytical figures of merit of homogeneous immunoassays with inductively coupled plasma mass spectrometry (ICP-MS) detection for multiplexed biomarker analysis in human serum samples. Specific monoclonal antibodies against four cancer biomarkers (CEA, sErbB2, CA 15.3 and CA 125) were labelled with different polymer-based lanthanide group to increase the number of metal labels per binding site. After the immunoreaction of the biomarkers with the specific antibodies, antigen-antibody complexes were separated by size-exclusion chromatography followed by ICP-MS detection. The polymer label could be loaded with 30-times more atoms of the lanthanide that the lanthanide-DOTA complex traditionally used for this purpose elsewhere [1] which resulted in a 10-fold improvement in both sensitivity and detection limits. Analytical figures of merit obtained with the lanthanide polymer labelling strategy make the detection of the biomarkers feasible below the threshold reference values used in clinical analysis. This labelling method was successfully validated by analyzing a control human serum spiked with the four biomarkers at three different concentration levels. For all the biomarkers studied, the recovery values ranged from 95% to 110% whereas inter-assay and intra-assay precision were lower than 8%. Results obtained with this approach were equivalent to those obtained by heterogenous-based immunoassays based on the detection by electro-chemiluminescence or ELISA. However, the method developed offers better analytical figures of merit using a smaller amount of sample. The authors would like to thank the Generalitat Valenciana (Project GV/2014/138) for the financial support of this work. E. Pérez also thanks the University of Alicante - Spain for the fellowship (UAFPU2011).

International audience; Uranyl ions (UO2 2+) were shown to interact with a number of foetal serum proteins, leading to the formation of a complex that could be isolated by ultracentrifugation. The molecular weight of the complex was estimated based on size-exclusion chromatography as 650 000 Da. Online ICP AES detection indicated that UO2 2+ in the complex co-eluted with minor amounts of calcium and phosphorous, but not with magnesium. A 1D gel electrophoresis of the U-complex produced more than 10 bands of similar intensity compared with only 2-3 intense bands corresponding to the main serum proteins in the control serum, indicative of the specific interaction of UO2 2+ with minor proteins. A proteomics approach allowed for the identification of 74 proteins in the complex. Analysis of the protein-protein interaction network in the UO2 2+ complex identified 32 proteins responsible for protein-protein complex formation and 34 with demonstrated ion-binding function, suggesting that UO2 2+ stimulates the formation of protein functional networks rather than using a particular molecule as its target. © 2015 The Royal Society of Chemistry.

International audience; A HPLC double reaction monitoring MS/MS method was developed for the determination of a wide range (>20) of sulphonamide residues in several edible animal (sheep, pork, beef, chicken and dromedary) tissues. Sample preparation was based on the simultaneous extraction into acetonitrile solution followed by a clean-up using primary secondary amine beads. Quantification was carried out using matrix-matched calibration curves. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.5 to 14.5 μg kg-1 and from 1.8 to 48.4 μg kg-1, respectively. The decision limit (CCα) and decision capability (CCβ) obtained were below 100 μg kg-1 for sulphonamides and below 5 μg kg-1 for dapsone. The method was validated in terms of recoveries and inter- and intra-day precision by reference analyses of meat samples using LC-Orbitrap MS and by the analysis of a reference material. The method was applied to the analysis of several animal tissue samples collected in Lebanon. The highest values were observed for sulfamethazine and sulfadimethoxine at 70.2 and 62.5 μg kg-1 in sheep tissues.

International audience; Glutathione peroxidase 1 (Gpx1), one of the most responsive selenoproteins to the variation of selenium concentration, is often used to evaluate ”selenium status” at a cellular or organismal level. The four major types of analytical methodologies to quantify Gpx1 were revisited. They include (i) an enzymatic assay, (ii, iii) polyacrylamide gel electrophoresis (PAGE) with (ii) western blot detection of protein or (iii) inductively coupled plasma mass spectrometry (ICP MS) detection of selenium, and (iv) size-exclusion chromatography with ICP MS detection. Each of the four methods was optimized for the quantification of Gpx1 with maximum sensitivity. The methods based on the enzymatic and immunodetection offer a much higher sensitivity but their accuracy is compromised by the limited selectivity and limited dynamic range. The advantages, drawbacks and sources of error of each technique are critically discussed and the need for the cross-validation of the results using the different techniques to assure the quality assurance of quantitative analysis is emphasized.

Selenium (Se) is an element readily absorbed during the intestinal tract and distributed in the body. In biological fluids, tissues, and animal products, Se is known to be present mainly in the form of a selenoamino-acid (selenomethionine (SeMet) or selenocysteine (SeCys)). Both amino-acids have different biological activities which justifies their discrimination. Here, we describe the method allowing the simultaneous determination of SeMet and SeCys in blood/plasma, animal tissues, milk, and eggs by two-dimensional Liquid Chromatography coupled to Inductively Coupled Plasma Mass Spectrometry (2D HPLC-ICP MS).

International audience; Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by μbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism. © 2014 Elsevier B.V. All rights reserved.

International audience; A proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS was developed to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast. Capillary HPLC-inductively coupled plasma mass spectrometry (ICP-MS), employed in parallel to capHPLC-ESI MS, indicated the virtual independence of the ESI MS response of the peptide structure (in the elution range of 30-65% methanol), and hence, the use of ESI MS data to determine the SeCys/Cys and SeMet/Met substitution ratios. For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. The SeMet/Met and SeCys/Cys ratios were determined in a large number of peptides (57 and 26, respectively) issued from the tryptic digestion of 19 Se-containing proteins located in the gel by laser ablation-ICP MS imaging. The average Se/S substitution in methionine was 42.9. ±. 35.0 and was protein dependent with ratios ranging from 5 to 160 for individual peptides. The substitution of sulphur in cysteine (14.1. ±. 4.8%) in the cysteine-containing peptides was relatively similar (ratios from 9 to 23). Taking into account that the cysteine/methionine average ratio (2:1) in the yeast protein fraction, the study allowed the conclusion that 10-15% of selenium present in Se-enriched yeast is in the form of selenocysteine making up the mass balance of selenium species. Biological significance: For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. It was achieved using a proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS in order to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast.

A matrix removal procedure with ion-exchange resin prior to analysis for 18 fluorinated benzoic acids (FBAs) tracers in saline (>25% salt) reservoir water was optimized. The elimination of >98% of salt and the simultaneous matrix sample cleanup allowed the direct analysis using the supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This resulted in a gain in detection limits for most of the tracers in comparison with the reference method (direct analysis after minimum required dilution). The limits of detection (LODs) were in the range of 0.01–0.15 ng/ml and compared to other studies the developed method provided comparable limits of detection and advantage of simplified and shorter sample preparation. The presented method offers a considerable gain in simplicity and analysis time. Recoveries for all the tracers reached 80–100%, except for 2-FBA and 2,6-dFBA for which they were ca. 60%. The low recoveries were corrected by the use of five isotopically labeled internal standards. The method was validated by the analysis of spiked samples and by an independent comparison of the results with those obtained by solid-phase extraction LC-MS/MS method. Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-0060-5) contains supplementary material, which is available to authorized users.

International audience; Gel permeation chromatography inductively coupled plasma high-resolution mass spectrometry (GPC ICP HR MS) was used for the understanding of the evolution of two crude oil cut samples after their dilution. We first studied different method parameters to compare two GPC procedures already published [flow rate, column set, and presence or not of the tetrahydrofuran (THF) stabilizer]. Thus, the principal parameters affecting the molecular size distribution and its evolution were demonstrated. The column set and flow rate can drastically affect the molecular size distribution of the sample. Moreover, an evolution of the size distribution of the complexes of vanadium, nickel, and sulfur into higher molecular weight compounds was observed over time. The study led to the recommendation of the sample preparation to the same day of the analysis to obtain reproducible data.

International audience; Description of metal species in plant fluids such as xylem, phloem or related saps remains a complex challenge usually addressed either by liquid chromatography-mass spectrometry, X-ray analysis or computational prediction. To date, none of these techniques has achieved a complete and true picture of metal-containing species in plant fluids, especially for the least concentrated complexes. Here, we present a generic analytical methodology for a large-scale (> 10 metals, > 50 metal complexes) detection, identification and semiquantitative determination of metal complexes in the xylem and embryo sac liquid of the green pea, Pisum sativum. The procedure is based on direct injection using hydrophilic interaction chromatography with dual detection by elemental (inductively coupled plasma mass spectrometry) and molecular (high-resolution electrospray mass spectrometry) mass spectrometric detection. Numerous and novel complexes of iron(II), iron(III), copper(II), zinc, manganese, cobalt(II), cobalt(III), magnesium, calcium, nickel and molybdenum(IV) with several ligands including nicotianamine, citrate, malate, histidine, glutamine, aspartic acid, asparagine, phenylalanine and others are observed in pea fluids and discussed. This methodology provides a large inventory of various types of metal complexes, which is a significant asset for future biochemical and genetic studies into metal transport/homeostasis.

International audience; A high efficiency chromatographic separation on a porous graphitic carbon stationary phase was developed for a large-scale separation of selenium metabolites in Se-rich yeast prior to their identification by electrospray hybrid quadrupole trap/Orbitrap mass spectrometry (Orbitrap MSn). The reversed-phase (RP) separation mode offered distinctly higher separation efficiency than the hydrophilic ion interaction (HILIC) mode. The latter was nevertheless complementary and useful to validate the detection of several compounds. The method allowed the detection of 64 metabolites including 30 SeSe or SeS conjugates (3 triple S/Se/S ones) and 14 selenoethers. 21 previously unreported metabolites were detected on the basis of the selenium isotopic pattern usually matched with the sub-ppm mass accuracy. 9 of these metabolites were subsequently identified using the multi-stage high mass accuracy (

International audience; A coupling of thin layer chromatography with laser ablation ICP SF MS was developed for the fractionation of Ni, V, Fe and S in crude oil and its fractions (saturate, aromatic, resin and asphaltene). The detection limits were 18 ng g -1 and 23 ng g -1 for nickel and vanadium, respectively, and a sample could be characterized in terms of the metal distribution as a function of species polarity within 10 min. The method was used to characterize the metal distribution in crude oils of different origins and their different fractions. © 2012 Elsevier B.V. All rights reserved.

International audience; Laser ablation-ICP MS was optimized for the sensitive detection of selenoproteins in polyacrylamide gel and PVDF membrane after blotting. For this purpose, two interlaboratory reference samples were prepared: glutathione peroxidase band in the gel and on the membrane, respectively. The optimisation was carried out using two systems: 213 nm laser (Newwave) - Agilent 7500ce ICP MS, and a 1030 nm high repetition rate femtosecond laser with galvanometric optics (Novalase) - PE/SCIEX DRCII. Sensitivity and signal-to-noise ratio were benchmarked to those obtained for the same sample by a recently published method in a reference lab. The optimization allowed a 12-fold gain of the S/N ratio during ablation of gels and a 3.5-fold gain in the ablation of blots in comparison with the method using an essentially similar system published by the reference lab. The gain of S/N by increasing ablation surface using the high repetition rate laser was not as spectacular as expected (2.5-fold for the gels and 1.5-fold for the blots) as the background noise increased considerably when a larger surface is ablated due to selenoproteins peak tailing. The study allowed for the first time LA-ICP MS detection of selenoproteins (separated by gel electrophoresis) in human cell extracts with the selenium concentration at the 10 ng ml -1 level.

International audience; A simple protocol to obtain Cu(II)-IDA (iminodiacetic acid)-modified capillaries was developed for immobilized metal ion affinity chromatography (IMAC). It consisted in the synthesis of IDA-silane used for a one-step coating of fused silica capillaries. The approach prevented the hydrolysis of silica potentially induced by two step coatings (γ-GPTMS, then IDA) employed in the conventional method of bonding iminodiacetic acid. The IDA content was quantified using a model relating the electroosmotic flow generated in IDA-modified capillaries to the charges induced by IDA species. The retention behavior of holotransferrin and bovine serum albumin on these IMAC columns was then investigated. Holotransferrin revealed a high affinity for Cu(II)-supports through a specific interaction with Cu(II) ions whereas albumin did not show any retention. The use of such columns for sample pretreatment before an HPLC analysis was proved to be successful. © 2011 Elsevier B.V. All rights reserved.

International audience; A method for the quantitation of proteinaceous selenocysteine (SeCys) in Se-rich yeast was developed. The method is based on the reduction of the Se-Se and S-Se bridges with dithiotretiol, derivatization with iodoacetamide (carbamidomethylation), followed by HPLC-ICP MS. The chromatographic conditions were optimized for the total recovery of the proteinaceous selenocysteine, the minimum number of peaks in the chromatogram (reduction of derivatization products of other Se-species present) and the baseline separation. A typical chromatogram of a proteolytic digest of selenized yeast protein consisted of up to five peaks (including SeMet, carbamidomethylated (CAM)-SeCys, and Se(CAM) 2) identified by retention time matching with available standards and electrospray MS. Inorganic selenium non-specifically attached to proteins and selenomethionine could be quantified (in the form of Se(CAM) 2) along with SeCys. Selenocysteine, selenomethionine, inorganic selenium, and the water soluble-metabolite fraction accounted for the totality of selenium species in Se-rich yeast.

International audience; Reasons for signal suppression during the analysis of light petroleum matrices by inductively coupled plasma mass spectrometry (ICP MS) were examined. A decrease of the ionization efficiency of the plasma was found to be the principal factor responsible for this loss of sensitivity. Consequently, an interface based on a total consumption micronebulizer and a heated spray chamber was constructed to alleviate this problem. A method based on flow-injection ICP MS using this interface was developed for the direct multielement analysis of undiluted fuels (gasoline, kerosene) and gas condensates offering an increase in sensitivity by at least a factor of 3-4 in comparison with the existing setups. © 2009 Elsevier B.V. All rights reserved.

International audience; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins. © 2008 Elsevier B.V. All rights reserved.

International audience; The reaction of sunflower oil with selenite produces a complex mixture of selenitriglycerides with antioxidant and anticancer properties. To obtain insight into the identity and characteristics of the species formed, an analytical approach based on the combination of high-performance liquid chromatography (HPLC) with 78 Se-specific selenium detection by inductively coupled plasma mass spectrometry (ICP MS) and high-resolution (100 000), high mass accuracy (

International audience; A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of watersoluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram level. The method, assisted by information obtained by MALDI TOF MS on the 5000 Da cut-off fraction, permitted the purity of bands and spots to be estimated and the efficiency of tryptic digestion and the quantity of selenium present in individual peptides to be evaluated. Owing to the high sensitivity and the lack of matrix suppression effects, the method provided chromatograms with signal-to-noise ratios of 10-1000 in conditions where the common ES Q-TOF MS detection failed.

International audience; Immunoaffinity chromatography has been investigated for fractionation of serum into selenoalbumin and true selenoproteins. Among several albumin-depletion kits tested, a multiaffinity column specifically binding albumin and five other major serum proteins provided the best results. It extracted ca 95% of both albumin and selenoalbumin, which enabled interference-free determination of glutathione peroxidase, selenoprotein P, and selenoalbumin by size-exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICPMS). The efficiency of the multiaffinity column did not vary over a period of 18 months. The purity of fractions separated by immunoaffinity LC was confirmed by elution-volume matching with standards in SEC-ICPMS and by selenopeptide mapping in capillary HPLC-ICPMS. Quantification of the selenium distribution among the different proteins in human serum from a control group and from a person on a selenium-rich diet revealed that 67% of the supplemented selenium was incorporated into albumin, 30% into glutathione peroxidase, and 3% into selenoprotein P.

cited By 14; International audience; Analysis of selenopeptides, isolated (and considered as pure) by 2D size-exclusion-reversed-phase HPLC, by capillary HPLC-ICP MS revealed a number of previously unreported Se-containing compounds. Their identity and origin were studied by the state-of-the art molecular mass spectrometric techniques including MALDI MS, oMALDI MS/MS and nanoLC-electrospray QTOF MS/MS. The research showed for the first time the possibility of loss of selenium from selenomethionine by degradation to vinylglycine and the formation of new Se peptides by methylation on carboxylic residues. The study substantiates claims for the use of the highest possible resolution HPLC for speciation studies. It raises concerns about misinterpretation of HPLC-ICP MS data on Se-speciation in selenised yeast since a number of peaks observed may be due to the formation of artefact compounds during some analytical procedures. © The Royal Society of Chemistry 2006.

International audience; The scope of the work was to investigate the influence of selenate fertilisation and the addition of symbiotic fungi (mycorrhiza) to soil on selenium and selenium species concentrations in garlic. The selenium species were extracted from garlic cultivated in experimental plots by proteolytic enzymes, which ensured liberation of selenium species contained in peptides or proteins. Separate extractions using an aqueous solution of enzyme-deactivating hydroxylamine hydrochloride counteracted the possible degradation of labile selenium species by enzymes (such as alliinase) that occur naturally in garlic. The selenium content in garlic, which was analysed by ICP-MS, showed that addition of mycorrhiza to the natural soil increased the selenium uptake by garlic tenfold to 15 μg g-1 (dry mass). Fertilisation with selenate and addition of mycorrhiza strongly increased the selenium content in garlic to around one part per thousand. The parallel analysis of the sample extracts by cation exchange and reversed-phase HPLC with ICP-MS detection showed that γ-glutamyl-Se-methyl-selenocysteine amounted to 2/3, whereas methylselenocysteine, selenomethionine and selenate each amounted to a few percent of the total chromatographed selenium in all garlic samples. Se-allyl-selenocysteine and Se-propyl-selenocysteine, which are selenium analogues of biologically active sulfur-containing amino acids known to occur in garlic, were searched for but not detected in any of the extracts. The amendment of soil by mycorrhiza and/or by selenate increased the content of selenium but not the distribution of detected selenium species in garlic. Finally, the use of two-dimensional HPLC (size exclusion followed by reversed-phase) allowed the structural characterisation of γ-glutamyl-Se- methyl-selenocysteine and γ-glutamyl-Se-methyl-selenomethionine in isolated chromatographic fractions by quadrupole time-of-flight mass spectrometry.

International audience; A solid-phase extraction (SPE) procedure using C18 stationary phase was optimized for the preconcentration of 19 fluorinated derivatives of benzoic acid (FBA): mono-, di-, tri-, and tetrafluorosubstituted in the ring, trifluoromethylbenzoic acid and 3,5-bistrifluoromethyl benzoic acid from undiluted salt-rich (>20%) reservoir waters. Quantitative (>90%) retention/elution of 16 out of 19 analyte compounds was achieved allowing a fourfold preconcentration factor accompanied by the elimination of >99% of salt. For the three most polar compounds (2,6-dFBA, 2,3,6-tFBA, and 2,4,6-tFBA) the non-quantitative recoveries (>70%) were corrected by dedicated custom-synthesized deuterated internal standards. The FBAs were determined by HPLC - MS/MS revisited in terms of a choice of column, elution conditions and MS/MS signal acquisition parameters allowing the baseline separation and a gain in sensitivity. For a sample intake of 4mL, detection limits for all the compounds in a reservoir water sample containing more than 20% salt were between 0.01 and 0.05ng/mL which represents a gain of a factor of 10-20 in comparison with the state-of the art LC-MS/MS procedures for samples of similar complexity. © 2015 Elsevier B.V.

The chapter, after summarizing the state-of-the art for selenium analysis and selenium status of different aliments, describes an analytical procedure that was developed for the quantitative recovery of selenomethionine (SeMet) and selenocysteine (SeCys) from foodstuffs of animal origin. It points out all the crucial steps of raw sample treatment and gives the details of all the steps that had to be optimized to achieve efficient protein extraction (protein unfolding/denaturation by urea) and ensure lack of losses (carbamidomethylation of the aminoacid residues using iodoacetamide) due to Se-compound degradation. The seleno-aminoacids were specifically determined by ion-paring reversed-phase HPLC-ICP MS and quantified by the method of standard addition after proteolysis followed by their isolation from the postreaction mixture by size-exclusion LC. In addition, formal identification by ESI MS/MS was carried out for compounds for which standards are not commercially available. Determination and quantification of separate selenoamino acids gives us information about specific or nonspecific incorporation of selenium into proteins.

International audience; A method allowing species-selective determination of atomic mercury, non-polar dialkylated mercury compounds, polar monoalkylated species and inorganic mercury complexes in natural gas condensates was developed. Inductively coupled plasma mass spectrometry was employed as a detection method for capillary gas chromatography and compared with microwave induced plasma atomic emission detection for the analysis of hydrocarbon-rich matrices. The method was based on two consecutive injections allowing comprehensive speciation analysis. First a sample aliquot was diluted with toluene and analysed for Hg0 and individual dialkylmercury compounds. Then, another aliquot was butylated with a Grignard reagent for the species specific determination of Hg(II) and monoalkylated mercury species. The detection limits were down to 0.08 pg level. © 2002 Elsevier Science B.V. All rights reserved.

International audience; Fifteen extraction methods were investigated for the recovery of different classes of Cd and Pb species in 8 different cocoa powder samples. The procedures targeted water-soluble compounds, polypeptide and polysaccharide complexes and compounds soluble in simulated gastrointestinal conditions. The extracts were analysed by size-exclusion fast-flow liquid chromatography with ICP-MS detection. The detection limit was 0.5 ?g l-1 and the RSD was less than 7.5%. Cd and Pb were very firmly bound to the insoluble matrix components, of which the binding capacity exceeded about 1000 times the naturally present metal levels. Cocoa powder may show possible detoxifying properties for Pb and Cd by binding them into stable complexes, which are resistant in gastrointestinal conditions. The maximum average recovery for Cd and Pb was, respectively, 15% and 5% of the total metal present.

International audience; This paper presents the design of an automated measurement device developed for speciation studies of organometallic compounds in samples of a complex and varying composition matrix. The instrument automates three essential steps in a typical speciation analysis procedure: isolation of analytes, their enrichment, and time-resolved introduction into a detector. Analytes are extracted by purging from post-sample dissolution/digestion mixture, in the form of volatile derivatives, with their simultaneous enrichment on a capillary cryotrap. Before introduction into a detector, they are rapidly (ca. 0.5 min.) separated on a multi-capillary gas chromatographic column operating in isothermal mode. The construction of the automated speciation analyser (ASA) makes it practically independent of a kind of the detector used (it was tested with MIP-AED,(1) ICP-MS,(2) and AAS detectors) and gives the freedom of choice of sample preparation procedure. The only requirement to the latter is that analytes should be in a form of volatile compound in an aqueous matrix.

On-line coupling of capillary GC with ICP-MS using the recently commercialized high-temperature interface was developed for the investigation of speciation of arsenic in gas condensates. Arsenic was present as a number of species, the two most abundant species being identified by retention time matching with standards such as triphenylarsine (Ph3As) and triethylarsine (Et3As). The calibration graph, obtained for Ph3As, was linear up to 100 ng mL−1 and the detection limit was 0.05 pg (as As). The arsenic speciation was found to undergo a slight evolution as a function of sample storage time. Spiking with standards was found to change the equilibrium in the system.

International audience; An off-gel non-denaturing isoelectric focusing (IEF) method was developed to separate uranium-biomolecule complexes from biological samples as a first step in a multidimensional metalloproteomic approach. Analysis of a synthetic uranium-bovine serum albumin complex demonstrated the focusing ability of the liquid-phase IEF method and the preservation of most of the uranium-protein interactions. The developed method was applied to gill cytosol prepared from zebrafish (Danio rerio) exposed to depleted uranium. The results were compared in terms of resolution, recovery, and protein identities with those obtained by in-gel IEF using an immobilized pH gradient gel strip. © 2014 Springer-Verlag Berlin Heidelberg.

International audience; Orthogonal liquid chromatographic (ion exchange, reversed phase, and ion pairing) and mass spectrometric [electrospray ionization (ESI)-TOF-MS, ESI-Orbitrap MS, and inductively coupled plasma mass spectrometry (ICP-MS)] methods were addressed to identify and quantify selenium species from a naturally Se-enriched green bean (Phaseolus vulgaris vulgaris) sample after proteolytic digestion. While selenomethionine (10.1 mg/kg as Se) and selenate (9.5 mg/kg as Se) could be quantified in a straightforward way by anion exchange LC-ICP-MS technique, a multistep purification protocol was required to identify Se-methylselenocysteine and γ-glutamyl-Se-methylselenocysteine in an unambiguous way prior to quantification by using either in-source fragmentation (LC-ESI-TOF-MS) or collision-induced dissociation (LC-ESI-Orbitrap MS). Finally, Se-methylselenocysteine (2.6 mg/kg as Se) and γ-glutamyl-Se-methylselenocysteine (1.2 mg/kg as Se) could contribute to the overall selenium recovery of 72 %. This sample is the first of the Faboideae subfamily and Phaseolus ssp. to be speciated to such an extent for selenium including γ-glutamyl-Se-methylselenocysteine, a highly potential selenium species, which makes this bean material an ideal candidate for functional food purposes. © 2013 Springer Science+Business Media New York.

International audience; A new high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method was developed for a simultaneous multi-residue analysis of 22 sulfonamides (SAs) and their metabolites in edible animal (pig, beef, sheep and chicken) tissues. Sample preparation was optimized on the basis of the "QuEChERS" protocol. The analytes were identified using their LC retention times and accurate mass; the identification was further confirmed by multi-stage high mass accuracy (

International audience; A semi-quantitative method was developed for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts without the use of radioactive isotopes. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL-1 (as Se). The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). Biological significance: Our paper presents the development of a semi-quantitative method for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts; it offers a first comprehensive screening of the entire biological selenoproteomes expressed in cell lines without the use of radioactive 75Se. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL-1 (as Se); by far the lowest ever reported. The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). The IEF-LA-ICPMS indicates the presence of multiple forms of some selenoproteins which are for the moment impossible to distinguish because of the similarity of the bottom-up, proteomics data sets.

International audience; The follow-up of the Heated Intraoperative Chemotherapy (HIPEC) of peritoneal carcinomatosis would benefit from the monitoring of the penetration, distribution and metabolism of the drug within the tumor. As tumor nodules can be resected during the therapy, mass spectrometry imaging is a suitable tool for the evaluation of treatment efficacy, and, as a result, the therapy can be re-optimized. In this work we demonstrate the complementarity of laser ablation (LA) ICP mass spectrometry and MALDI imaging to study the penetration and distribution of two Pt-based metallodrugs (cisplatin and oxaliplatin) in human tumor samples removed from patients diagnosed with colorectal or ovarian peritoneal carcinomatosis. LA ICP MS offered sensitive (LOD for 195Pt 4.8 pg s-1) imaging of platinum quasi-independently of the original species and the sample matrix and thus an ultimate way of verifying the penetration of the Pt-containing drug or its moieties into the tumor. MALDI imaging was found to suffer in some cases from signal suppression by the matrix leading to false negatives. In the case of the oxaliplatin metallodrug, the results obtained from ICP and MALDI MS imaging were coherent whereas in the case of cisplatin, species detected by ICP MS imaging could not be validated by MALDI MS. The study is the first application of the dual ICP and MALDI MS imaging to the follow-up of metallodrugs in human tumors

International audience; A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4 ± 0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03 ± 0.23, 15.27 ± 0.36, and 177.31 ± 25.51 nmol U L-1 in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L-1 during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L-1. Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (μRPC-ESI-MS/MS) after ND-IEF separation. [Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg.

International audience; A method was developed for the quantification of Zn-, Cu- and Mn-glycinates in supplemented feed samples. The coupling of capillary electrophoresis (CE) with ICP MS detection after purification of the extract by ultrafiltration was shown to be efficient for the quantitative recovery of glycinates. The method developed was then applied to evaluate the bioaccessibility of glycinates using a sequential enzymolysis approach. The data obtained indicated a strong bioaccessibility of each element (79-94%). A new complex was also found to be formed during the digestion process. Bioavailability was then evaluated by analyzing plasma samples of horses supplemented with glycinates-rich feed. Intact glycinates could not be detected in plasma samples but a Cu-containing molecule was found more abundant after CuGly treatment. © 2013 Elsevier B.V.

International audience; The identification of the ligands binding Al is essential to understand the mechanisms by which plants detoxify Al internally. However, studies concerning the speciation of Al have been frustrated by its complex chemistry. This work describes the identification of the tri-Al tricitrate (Al3cit 3) complex in Plantago almogravensis, encompassing an integrated mass spectrometry approach based on hydrophilic interaction liquid chromatography (HILIC) and parallel detection by ICP-MS and ESI-MS/MS. This work also reports that both Al and Fe are bound by tricitrate, sometimes simultaneously, and the consequences of this finding are discussed. Of the complexes separated by size exclusion chromatography, Al3cit3 is the most stable occurring in P. almogravensis as it was the only one recovered after HILIC. This approach provided new information on the mechanism of Al detoxification in P. almogravensis, namely that Al is bound by the organic acid citrate and that the relative concentration of the detected complexes is affected by the organ type and internal Al concentration, and has potential for studying the speciation of Al in less tolerant plants. © 2013 The Royal Society of Chemistry.

International audience; A novel analytical procedure for the identification of metal (Hg, Cd, Cu, Zn) complexes with individual metallothionein (MT) isoforms in biological tissues by electrospray MS/MS was developed. The sample preparation was reduced to three rapid steps: the two-fold dilution of the sample cytosol with acetonitrile, the recovery of the supernatant containing MT-complexes by centrifugation and its concentration under nitrogen flow. The replacement of reversed phase HPLC by hydrophilic interaction LC (HILIC) allowed the preservation of the unstable and low abundant metallothionein zinc-mercury mixed complexes (MT-Zn6Hg). The MT complexes eluted were detected by ICP MS and identified in terms of molecular mass by electrospray high resolution (100000) MS. The identification was completed by on line demetallation and the determination of the molecular mass of the apoform, followed by amino acid sequencing in the top-down mode using high energy collision fragmentation (HCD). The method was applied to the identification of MT complexes in a white-sided dolphin (Lagenorhynchus acutus) liver homogenate. The Zn complex of the N-acetylated MT2 isoform was found to be predominant, the presence of mixed complexes with Cd, Cu and, for the first time ever, Hg, was demonstrated. The latter finding has the potential to shed new light on the mercury detoxification mechanism in marine organisms.