Determination of Proteinaceous Selenocysteine in Selenized Yeast

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

Analytical techniques for speciation of selenium in food and food supplements: State-of-the-art

DOI: 10.3920/978-90-8686-642-7International audienceno abstrac

Accumulation of As, Ag, Cd, Cu, Pb, and Zn by Native Plants Growing in Soils Contaminated by Mining Environmental Liabilities in the Peruvian Andes

International audienceThe capability of native plant species grown in polluted post-mining soils to accumulate metals was evaluated in view of their possible suitability for phytoremediation. The study areas included two environmental liabilities in the Cajamarca region in the Peruvian Andes. The content of As, Ag, Cd, Cu, Pb, and Zn was determined in individual plant organs and correlated with soil characteristics. The degree of the pollution depended on the metal with results ranging from uncontaminated (Cd) to moderately (Zn), strongly (As, Cu), and extremely contaminated (Pb, Ag) soils. The metals were mainly present in the fractions with limited metal mobility. The bioaccumulation of the metals in plants as well the translocation into overground organs was determined. Out of the 21 plants evaluated, Pernettya prostrata and Gaultheria glomerate were suitable for Zn, and Gaultheria glomerata and Festuca sp. for Cd, phytostabilization. The native species applicable for Cd hytoremediation were Ageratina glechonophylla, Bejaria sp., whereas Pernettya prostrata Achyrocline alata, Ageratina fastigiate, Baccharis alnifolia, Calceolaria tetragona, Arenaria digyna, Hypericum laricifolium, Brachyotum radula, and Nicotiana thyrsiflora were suitable for both Cd and Zn. None of the studied plants appeared to be suitable for phytoremediation of Pb, Cu, As and Ag

Identification and determination of selenohomolanthionine – The major selenium compound in Torula yeast

International audienceTorula yeast (Candida utilis) was found to metabolize selenium in a totally different way to Brewer's yeast (S. cerevisiae) leading to the biosynthesis of selenohomolanthionine (SeHLan), a major selenium compound accounting for 60-80% of the total selenium. The identity of SeHLan was confirmed by retention time matching in hydrophilic ion interaction chromatography (HILIC) with inductively coupled plasma mass spectrometric detection (ICP MS) using a custom synthesized standard molecule and by HILIC-Orbitrap MS and MS-MS fragmentation. Selenohomolanthionine escapes the current assays for the organic character of Se-rich yeast based on the protein-bound selenomethionine determination. A HILIC-ICP MS method was developed for the quantitative determination of selenohomolanthionine in yeast supplements with a detection limit of 146 ng/g

Speciation of Selenium in Selenium-Enriched Sunflower Oil by High-Performance Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry/Electrospray–Orbitrap Tandem Mass Spectrometry

International audienceThe 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 78Se-specific selenium detection by inductively coupled plasma mass spectrometry (ICP MS) and high-resolution (100 000), high mass accuracy (<1 ppm) molecule-specific detection by electrospray–Orbitrap MS3 was developed. For the first time, a non-aqueous mobile phase gradient was used in reversed-phase HPLC–ICP MS for the separation of a complex mixture of selenospecies and a mathematical correction of the background signal was developed. The identical chromatographic conditions served for the sample introduction into electrospray MS. Two types of samples were analyzed: sunflower oil dissolved in isopropanol and methanol extract of the oil containing 65% selenium. HPLC–ICP MS showed 14 peaks, 11 of which could also be detected in the methanol extract. Isotopic patterns corresponding to molecules with one or two selenium atoms could be attributed by Orbitrap MS at the retention times corresponding to the HPLC–ICP MS peak apexes. Structural data for these species were acquired by MS2 and MS3 fragmentation of protonated or sodiated ions using high-energy collisional dissociation (HCD). A total of 11 selenium-containing triglycerol derivatives resulting from the oxidation of one or two double bonds of linoleic acid and analogous derivatives of glycerol-mixed linoleate(s)/oleinate(s) have been identified for the first time. The presence of these species was confirmed by the targeted analysis in the total oil isopropanol solution. Their identification corroborated the predicted elution order in reversed-phase chromatography: LLL (glycerol trilinoleate), LLO (glycerol dilinoleate–oleinate), LOO (glycerol linoleate–dioleinate), OOO (glycerol trioleinate), of which the extrapolation allowed for the prediction of the identity [glycerol dioleinate–stearate (OOS) and glycerol oleinate–distearate (OSS)] of the nonpolar species detected by ICP MS in the oil but not detected by electrospray MS

Screening for polybrominated diphenyl ethers in biological samples by reversed-phase fast HPLC-ICP MS

International audienceThe coupling of fast (ultrahigh pressure) liquid chromatography with ICP MS fitted with an octopole collision cell was developed for the separation and detection of ten polybrominated diphenyl ethers (PBDE) (47, 85, 100, 138, 155, 201, 206, 207, 208, 209). The compounds were eluted within 12 min using an acetonitrile-rich (70-95%) mobile phase at 1.5 mL min-1 which could be introduced directly into an inductively coupled plasma owing to a new plasma frequency-marching RF generator. The limit of detection (as bromine) at these conditions was 17 ± 1 ng mL-1 and was independent of the mobile phase composition. The method was applied to the screening metabolites of the high molecular weight PBDE decabromodiphenyl ether in rat liver and faeces and was validated by independent HPLC with radioactivity detection. It offered, as a main advantage over molecular MS, the virtual independence of the signal intensity of the molecular structure of the compound, and thus the possibility to detect unknown metabolites and control bromine mass balance

A comparative study of the Se/S substitution in methionine and cysteine in Se-enriched yeast using an inductively coupled plasma mass spectrometry (ICP MS)-assisted proteomics approach

International audienceA 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