Véronique Vacchina

An approach to the identification of selenium species in yeast extracts using pneumatically-assisted electrospray tandem mass spectrometry

An approach to the identification of unknown signals in selenium speciation analysis of yeast by reversed-phase chromatography with ICP-MS detection is described. The analytical strategy was based on: (i), heart-cutting of a Se-containing fraction in the reversed-phase chromatographic eluate followed by its lyophilization; (ii), pneumatically-assisted electrospray (ESI) MS and ESI tandem MS of the lyophilizate; and (iii) confirmation of the fragmentation pattern obtained using the sulfur analogue of the seleno compound that was expected to have been identified. The approach developed allowed the identification of Se–adenosylhomocysteine as the major selenium species in an extract of a selenized yeast sample.

Speciation of cadmium in plant tissues by size-exclusion chromatography with ICP-MS detection

A systematic approach based on the coupling of high resolution size-exclusion chromatography (SEC) with ICP-MS detection was developed for the speciation of Cd complexes with oligopeptides known to be biosynthesized by plants exposed to metal stress (phytochelatins, PCs). The separation conditions were optimized using a series of standards prepared by incubation of cadmium with purified (GluCys) 2 Gly (PC 2 ), (GluCys) 3 Gly (PC 3 ) and (GluCys) 4 Gly (PC 4 ) ligands. The formation of artefact species was investigated and conditions allowing the reproducible chromatography and quantitative determination of Cd species in the range 0.01-15 µg ml –1 were established. The method developed was applied to investigate the speciation of Cd in cytosols of plant tissues and plant cell cultures. In addition to Cd complexes with PC 2 , PC 3 and PC 4 that eluted at the times corresponding to standards, two other Cd species, one excluded from the column and the other with an apparent molecular mass of 6800 Da, were observed in all the samples investigated. The latter compound, isolated and analysed by electrospray MS-MS, turned out to be a mixed Cd complex with different PC ligands. The species eluted in the void volume could not be identified. The method was validated by comparing the results obtained by SEC-ICP-MS with those obtained by reversed-phase HPLC with post-column derivatization of phytochelatin.

Methodological advances for selenium speciation analysis in yeast

Abstract Advances in analytical methodology for speciation of selenium in selenized-yeast food supplements were discussed on the basis of the recent developments in the authors’ laboratory. Particular attention was given to the sample preparation with regard to the fractionation of selenium into different classes of chemical species, the high resolution fractionation of selenium from yeast water extracts by size-exclusion chromatography and characterization of the water soluble protein fraction by combined matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry (TOF MS) and electrospray quadrupole-TOF tandem MS. The true speciation of protein-incorporated selenium (down to individual proteins characterized by a unique aminoacid sequence) was discussed using an example of a family of selenium-containing proteins formed in yeast by the substitution of methionine residues by selenomethionine in a salt stress-induced protein.

Identification of phytochelatin-related peptides in maize seedlings exposed to cadmium and obtained enzymatically in vitro

An analytical strategy based on the sensitivity of electrospray tandem mass spectrometry following a simplified and reproducible sample preparation procedure was evaluated for the determination of Cd-induced phytochelatins (PC) and related peptides in four maize varieties. In addition to the three known families of PC (PC, desGly-PC and iso-PC(Glu)) that were observed, novel PC and desGly-PC homologues lacking the N-terminal gamma-linked Glu were isolated from maize root extracts for the first time. Additionally the complete sequence of iso-PC3(Glu) was determined by tandem mass spectrometry. Peptides obtained in vivo and in vitro as the result of the reaction of glutathione with the enzyme phytochelatin synthase were compared. Minor forms detected from in vitro reactions include compounds with intramolecular or intermolecular disulfide bonds resulting from the oxidation of SH groups, phytochelatin homologues lacking the N-terminal gamma-linked Glu, and new PC-related peptides with a Cys-Cys motif. Since peptides lacking a gammaGlu residue could be generated as artifacts in electrospray mass spectrometry, the application of capillary electrophoresis with online electrospray mass spectrometry allowed the separation and detection of such peptides as endogenous molecules present in planta and as products of in vitro reactions.

Elemental speciation analysis in biochemistry by electrospray mass spectrometry

Abstract The recent advances and trends in the development and applications of electrospray mass spectrometry (ESI-MS) to speciation analysis are highlighted. The use of the different operational modes of ESI-MS: elemental mode (source collision-induced dissociation (SCID)), molecular mode (MS of protonated molecule ions) and tandem MS mode (product ion scan of the collision-induced dissociation (CID) products of the protonated molecule ion), is discussed. Advantages and limitations of ESI-MS for signal identification in high performance liquid chromatography (HPLC)–ICP-MS and as a free-standing tool for species-selective analysis are highlighted. The discussion is illustrated with a number of examples related to the trace element speciation in biochemistry, viz. selenium in yeast, arsenic in seaweeds, cobalamins in pharmaceutical preparations, and Cd complexes with phytochelatins and metallothioneins in plant and animal tissues

Signal identification in size-exclusion HPLC-ICP-MS chromatograms of plant extracts by electrospray tandem mass spectrometry (ES MS/MS)

Pneumatically-assisted electrospray (ion-spray®) tandem mass spectrometry (ES-MS/MS) was evaluated for the identification of cadmium binding ligands in complexes detected in extracts of plant cell cultures by size-exclusion HPLC (SEHPLC) with ICP-MS detection. The metal-containing fractions were heartcut, purified by reversed-phase chromatography, and analysed by ES-MS/MS. The purity and identity of signals in ES-MS/MS spectra were further confirmed by collision induced dissociation (CID)-MS. The different peaks in SEHPLC-ICP-MS turned out to contain identical ligands at different ratios. On the other hand, a single SEHPLC-ICP-MS chromatographic signal was demonstrated to contain a number of complexes with ligands varying in terms of amino-acid chain length, amino-acid sequence and stoichiometry. Whereas the particular ligands (phytochelatins modified on the C-terminal amino-acid) could be identified, the complexity of equilibria within the isolated fraction prevented the elucidation of the stoichiometry of the complexes formed. The results question the validity of signal identification in SEHPLC-ICP-MS by elution volume matching with standards unless the chromatographic purity of peaks is demonstrated.

Characterisation and determination of phytochelatins in plant extracts by electrospray tandem mass spectrometry

A method based on pneumatically assisted electrospray ionisation tandem mass spectrometry (ESI MS-MS) was developed for the identification, sequencing and determination of phytochelatin (PC) peptides in plant tissue and plant cell cytosols. The ionization and fragmentation conditions were optimized using a series of (GluCys)2Gly (PC2), (GluCys)3Gly (PC3), and (GluCys)4Gly (PC4) standards prepared from glutathione by enzymatically (γ-glutamylcysteine dipeptyl transpeptidase) assisted biosynthesis in the presence of Cd2+. Phytochelatins were found to ionize readily to produce a characteristic mono-protonated ion. The collision-induced dissociation (CID) of this ion followed by mass spectrometry (MS-MS mode) allowed the determination of the amino acid sequence of each of the PCs. Calibration curves were linear up to a concentration of 2 µg ml–1 in the MS and MS-MS modes with the detection limits at the low ng ml–1 level. The method was applied to the determination of phytochelatin peptides biosynthesized by a number of plant cell cultures exposed to the Cd stress. The results agreed with those obtained by an independent procedure based on reversed-phase HPLC with post-column derivatization of the –SH groups with 5,5′-dithiobis-2-nitrobenzoic acid and spectrophotometric detection.

Gas and liquid chromatography with inductively coupled plasma mass spectrometry detection for environmental speciation analysis — advances and limitations☆

Abstract Recent advances in the coupling of gas chromatography (GC) and high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP MS) and their role in trace element speciation analysis of environmental materials are presented. The discussion is illustrated with three research examples concerning the following topics: (i) development and coupling of multicapillary microcolumn GC with ICP MS for speciation of organotin in sediment and biological tissue samples; (ii) speciation of arsenic in marine algae by size-exclusion-anion-exchange HPLC-ICP MS; and (iii) speciation of cadmium in plant cell cultures by size-exclusion HPLC-ICP MS. Particular attention is paid to the problem of signal identification in ICP MS chromatograms; the potential of electrospray MS/MS for this purpose is highlighted.

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.

Determination of phytochelatins by capillary zone electrophoresis with electrospray tandem mass spectrometry detection (CZE-ES MS/MS)

The coupling of capillary zone electrophoresis with electrospray mass spectrometry was optimized for the direct determination of phytochelatins (PCs) in extracts obtained from cells and plants that had been exposed to metal stress. Gluthathione and phytochelatins belonging to the different families (gamma Glu-Cys)nGly (n-PC), (gamma Glu-Cys)nSer, (gamma Glu-Cys)n beta Ala and (gamma Glu-Cys)n were separated in an uncoated capillary at pH 4 using a 5 mM ammonium acetate buffer, and detected by electrospray (ES) MS in the full scan mode (300-1100 u). The use of on-line tandem MS detection in the product ion scan mode of putative protonated molecules of PCs allowed the unambiguous confirmation of the identity of the compounds detected by ES MS. The operational conditions were optimized and the figures of merit were evaluated using n-PC2, n-PC3 and n-PC4 standards purified from a mixture obtained after the reaction of glutathione in the presence of Cd2+ and the enzyme PC-synthase. The method was applied to the characterization of bioinduced ligands in cell cultures of soybeans (Glycine max) and in rice (Oryza sativa) roots without the need for a preliminary sample cleanup by size-exclusion and/or reversed phase chromatography.