Katarzyna Bierla

Selenium metabolomics in yeast using complementary reversed-phase/hydrophilic ion interaction (HILIC) liquid chromatography–electrospray hybrid quadrupole trap/Orbitrap mass spectrometry

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 MS(n)). 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 (<5ppm) mass spectrometry. The identified metabolites (and their groups) were quantified on-line by ICP-MS fitted with a frequency-matching generator allowing a quasi-uniform response over the large (20-90%) acetonitrile mobile phase concentration range. The morphology of HPLC-ICP-MS chromatograms was remarkably similar to that of HPLC multi-ion extracted ESI-MS chromatograms. The detection limits obtained by ICP MS and ESI MS were 1 and 2ppb, respectively.

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

UNLABELLED 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.

Study of the Se-containing metabolomes in Se-rich yeast by size-exclusion—cation-exchange HPLC with the parallel ICP MS and electrospray orbital ion trap detection

Strong cation exchange HPLC with the parallel ICP MS and electrospray hybrid linear ion trap quadrupole orbital trap mass spectrometry (ESI Orbitrap MS) detection was developed for the study of the metabolomic pattern of selenium in selenium-rich yeast. The mobile phase composition (gradient of ammonium formate in 20% methanol) was optimized to obtain separation in conditions guaranteeing the identical ICP MS sensitivity during the entire chromatographic run and the compatibility with electrospray ionization. Twenty seven Se-containing metabolites observed in the HPLC-ICP MS chromatogram were identified by ESI Orbitrap MS based on the Se isotopic pattern, the accurate molecular mass, and the multistage fragmentation patterns. The method allowed for the first time the correlation of the differences observed in HPLC-ICP MS chromatography of water extracts of Se-rich yeast samples from different manufacturers with the identity of the eluted compounds determined by ESI MS.

A sequential extraction procedure for an insight into selenium speciation in garlic.

A sequential extraction procedure was developed for the fractionation of different classes of selenium species present in garlic. The consecutive steps included leaching with water, extraction of cell-wall bound species after lysis with a mixture of cellulase, chitinase and beta-glucanase completed by a proteolytic attack, extraction with HCl to liberate the residual organic bound species and finally, extractions with sulfite solution and CS(2) to complete the mass balance by the recovery of Se(0) and Se(2-), respectively. Selenium speciation in the aqueous fractions was probed by anion-exchange and ion-pairing reversed-phase HPLC-ICP MS after purification by preparative size-exclusion LC. It was found to be strongly affected by the sample redox conditions. The peak identity was matched with a mixture of 9 compounds expected to be present in allium plants; electrospray QTOF MS turned out to be unsuccessful. Selenite, selenate and selenomethionine were the dominating species present.

Simultaneous derivatization of selenocysteine and selenomethionine in animal blood prior to their specific determination by 2D size-exclusion ion-pairing reversed-phase HPLC-ICP MS

A procedure for the simultaneous quantitative carbamidomethylation of selenocysteine (SeCys) and selenomethionine (SeMet) followed by their proteolytic release from blood proteins was developed. The fraction containing both derivatized selenoaminoacids was isolated by size-exclusion chromatography and submitted to ion-pairing HPLC-ICP MS analysis. The limit of detection was ca. 0.02 μg g−1 (dry mass) for either amino acid. The quantification of SeCys and SeMet was carried out by the method of standard additions. An internal standard of 77Se-labelled SeMet was used to control the derivatization yield and chromatographic recovery. The determination of SeCys was validated by spiking with glutathione peroxidase. An additional proof of validity was achieved by monitoring the selenium mass balance (12 series of analysis over a period of 18 months; the Se amino acids accounted for 92 ± 8% of the total Se). The method was applied to the monitoring of changes in SeCys and SeMet concentrations in lamb blood during supplementation studies (tolerance and dose effect) with selenium-rich yeast.

Specific determination of selenoaminoacids in whole milk by 2D size-exclusion-ion-paring reversed phase high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP MS)

A procedure was developed for the quantitative recovery of selenomethionine (SeMet) and selenocysteine (SeCys) from whole milk. It was based on the protein unfolding, carbamidomethylation of the aminoacid residues using iodoacetamide and proteolysis using Protease XIV. The selenoaminoacids were specifically determined by ion-paring reversed phase HPLC-ICP MS after their isolation from the post-reaction mixture by size-exclusion LC. Se(IV) present in the sample was derivatized as well and was determined along with the selenoaminoacids. The origin and identity of species were identified by the co-elution with the Se(IV), isotopically labelled selenomethionine, and with the synthetic standard of carbamidomethylated selenocysteine. The method development for SeCys was assisted by using glutathione peroxidase as the SeCys standard. SeMet, SeCys and Se(IV) were quantified by the method of standard additions. The mass balance provided a measure of the method validation. The method was applied to monitoring selenium speciation during supplementation of cows (dose-effect study) with Se-rich yeast containing feed and during milk processing.

Comprehensive speciation of low-molecular weight selenium metabolites in mustard seeds using HPLC – electrospray linear trap/orbitrap tandem mass spectrometry

An analytical methodology based on high-resolution high mass accuracy electrospray ionization (ESI) tandem MS assisted by Se-specific detection using inductively coupled plasma mass spectrometry (ICP MS) was developed for speciation of selenium (Se) in seeds of black mustard (Brassica nigra) grown on Se-rich soil. Size-exclusion LC-ICP MS allowed the determination of the Se distribution according to the molecular mass and the control of the species stability during extraction. The optimization of hydrophilic interaction of LC and cation-exchange HPLC resulted in analytical conditions making it possible to detect and characterize over 30 Se species using ESI MS, including a number of minor (<0.5%) metabolites. Selenoglucosinolates were found to be the most important class of species accounting for at least 15% of the total Se present and over 50% of all the metabolites. They were found particularly unstable during aqueous extraction leading to the loss of Se by volatilization as methylselenonitriles and methylselenoisothiocyanates identified using gas chromatography (GC) with the parallel ICP MS and atmospheric pressure chemical ionization (APCI) MS/MS detection. However, selenoglucosinolates could be efficiently recovered by extraction with 70% methanol. Other classes of identified species included selenoamino acids, selenosugars, selenosinapine and selenourea derivatives. The three types of reactions leading to the formation of selenometabolites were: the Se-S substitution in the metabolic pathway, oxidative reactions of -SeH groups with endogenous biomolecules, and chemical reactions, e.g., esterification, of Se-containing molecules and other biomolecules through functional groups not involving Se.

Speciation analysis for trace levels of selenoproteins in cultured human cells

UNLABELLED 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 (75)Se. 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.

Bioaccessibility of Se from Se-enriched wheat and chicken meat.

Selenium (Se) is an essential trace element to animals and humans as Se is incorporated in a series of organic molecules, such as 30 mammalian selenoproteins or seleno-enzymes, which are vital for the basic functions of life. To increase the Se intake in Se-deficient areas, food and feed can be enriched using Se fertilizers or supplements. The aim of this study is to investigate the distribution, speciation, bioaccessibility, and bioavailability of Se in Se-enriched wheat (SW) grain and in Se-enriched chicken meat products using commercial enzymes and human gastric juices (HGJs). Results from the present work show that Se in wheat is bioaccessible and bioavailable, and that SW flour or bran can serve as a valuable dietary source of Se to humans. However, the bioaccessibility studies using commercial enzymes and HGJs for wheat flour, bran, and chicken meat digestion suggests that the use of commercial enzymes overestimate Se bioavailability. Furthermore, the use of NaCl or Tris-HCl to extract Se proteins from enriched products was not suited for bioaccessibility studies. The SW flour or bran can, however, serve as a valuable dietary source of Se to humans.

Accurate determination of selenium in blood serum by isotope dilution analysis using inductively coupled plasma collision cell mass spectrometry with xenon as collision gas

A method for accurate selenium determination in blood serum by isotope dilution analysis (IDA) was developed. Selenium was determined by ICP octopole reaction system MS using xenon as the collision gas (130–160 μL min−1) in order to eliminate polyatomic interferences from argon, chlorine, phosphorus and bromine. The detection limit was 3.3 μg L−1, which was higher than with hydrogen (0.4 μg L−1) but sufficiently sensitive to determine selenium in serum. Selenium isotope ratios could be determined precisely and accurately without any mathematical interference corrections, enabling IDA at the 80Se/76Se ratio. In contrast, these corrections were necessary when hydrogen was used as the reaction gas due to the formation of SeH+ (2.4%) and BrH+ (14%), but even then systematic errors in the correction of the BrH+ interference could not be avoided. Hence, the selenium recovery from human serum reference material was only 78.0 ± 0.4% when measured with hydrogen but 96.7 ± 4.0% with xenon.