Use of metallomics approach to the bivalve mollusc Chamaelea gallina by SEC-UV-ICP MS followed by RP-HPLC-ICP MS

Use of metallomics approach to the bivalve mollusc Chamaelea gallina by SEC-UV-ICP MS followed by RP-HPLC-ICP MS

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čeština

The aim of the present study is the characterization of metals bound to biomolecules in the bivalve Chamalea gallina, from the Andalusian coast (southwest Spain). This area is affected by the action of metal pollution caused by mining activities, which fact could modify the biomolecules expression of this bivalve.Total content of elements was determined by ICP MS, which revealed a remarked presence of Fe, Mn, Cu, Zn, and As in the samples. Therefore, further studies were focused on these elements. In the metallomics study extracts of bivalves were prepared from whole bodies of clams that were grounded in a mortar with liquid nitrogen, and subsequently homogenized with an Ultraturrax apparatus and stored freezed until analys. Portions of freezed sample were weighed and three times extracted with water solution of phenylmethylsulfonyl fluoride and dithiotreithol in an ultrasonic bath. The extracts were centrifuged, and the combined extracts were lyophilized and store freezed no longer than two weeks. An aliquot of the extract was dissolved in water and injected onto the preparative size exclusion column Superdex 30 pg HiLoad 26/60 and eluted with 0,05 M phosphate buffer in 0,15 M sodium chloride (pH 7,2) at 2 ml/min. The column was coupled to a ICP-MS for metal detection. At least four fractions molecular weight in the range 1600 to 700 Da was observed with UV detection, but the ICP-MS chromatogram showed the co-elution of metals of interest only in the two earlier fractions. The apparent MWs of these metals-containing fractions were between 1525-950 Da, as evaluated from the column calibration. The fractions cointaining metals compounds were collected and then lyophylized. This lyophilizate was dissolved in water and submitted to a second reverse phase (C18) chromatographic separation with ICP-MS detection using 0,1 % TFA gradient in methanol at the flow rate of 0,75 ml/min. Several peaks were obtained for metals of interest. RP-HPLC was repeated and fractions containing metals compounds were collected and then lyophilized. Finally, the lyophilizate from a RP-HPLC dissolved in 50 % methanol was studied by ESI-Q-TOF MS/MS for biomolecule identification.

The aim of the present study is the characterization of metals bound to biomolecules in the bivalve Chamalea gallina, from the Andalusian coast (southwest Spain). This area is affected by the action of metal pollution caused by mining activities, which fact could modify the biomolecules expression of this bivalve.Total content of elements was determined by ICP MS, which revealed a remarked presence of Fe, Mn, Cu, Zn, and As in the samples. Therefore, further studies were focused on these elements. In the metallomics study extracts of bivalves were prepared from whole bodies of clams that were grounded in a mortar with liquid nitrogen, and subsequently homogenized with an Ultraturrax apparatus and stored freezed until analys. Portions of freezed sample were weighed and three times extracted with water solution of phenylmethylsulfonyl fluoride and dithiotreithol in an ultrasonic bath. The extracts were centrifuged, and the combined extracts were lyophilized and store freezed no longer than two weeks. An aliquot of the extract was dissolved in water and injected onto the preparative size exclusion column Superdex 30 pg HiLoad 26/60 and eluted with 0,05 M phosphate buffer in 0,15 M sodium chloride (pH 7,2) at 2 ml/min. The column was coupled to a ICP-MS for metal detection. At least four fractions molecular weight in the range 1600 to 700 Da was observed with UV detection, but the ICP-MS chromatogram showed the co-elution of metals of interest only in the two earlier fractions. The apparent MWs of these metals-containing fractions were between 1525-950 Da, as evaluated from the column calibration. The fractions cointaining metals compounds were collected and then lyophylized. This lyophilizate was dissolved in water and submitted to a second reverse phase (C18) chromatographic separation with ICP-MS detection using 0,1 % TFA gradient in methanol at the flow rate of 0,75 ml/min. Several peaks were obtained for metals of interest. RP-HPLC was repeated and fractions containing metals compounds were collected and then lyophilized. Finally, the lyophilizate from a RP-HPLC dissolved in 50 % methanol was studied by ESI-Q-TOF MS/MS for biomolecule identification.

metallomics, Chamaelea gallina, SEC, HPLC, ICP-MS

VITOULOVÁ, E.; GÓMEZ-ARIZA, J.; GARCÍA-BARRERA, T.; FIŠERA, M.

27. 8. 2006

17 th IMSC Secretariat

Praha

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