17th International Mass Spectrometry Conference :: Prague, 2006
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|Session:||Food and Nutrition|
|Presentation date:||Mon, Aug 28, 2006|
|Presentation time:||14:30 – 16:00|
Eva Vitoulova1, Jose Luis Gomez Ariza2, Miroslav Fisera11 Brno University of Technology, Faculty of Chemistry, Brno, Czech Republic
Correspondence address: Eva Vitoulova, Brno University of Technology, Faculty of Chemistry, Institute of Food Science and Biotechnology, Purkynova 118, Brno, 61200 Czech Republic.
Keywords: Mass Spectrometry, Inductively Coupled Plasma; Mass Spectrometry, Liquid Chromatography; Metalloprotein; Metals.
Novel aspect: Metallomics study of metal-biomolecules.
Chamaelea gallina has been used sentinel organism in the study of the biological effects of pollutants. Several proteins have been proposed as biomarkers, especially oxidative enzymes and metallothioneins.1 However, more of these studies used traditional proteomics approaches based in (2-D) electrophoresis and proteins identification by mass spectrometry. However, more recent studies focused on the combined use of both metal detector (ICP-MS) and molecule detectors (MALDI-TOF and Q-TOF) for rapid and unequivocal identification of metal-biomolecules (metallomics)2,3 have not been considered.
The aim of the present study is the characterization of metals bound to biomolecules in the bivalve Chamaelea 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 at -20°C until analyse. 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 stored at -20°C 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.
1. J. L. Lopez-Barea et al., Proteomics 3, 1535 (2003).
2. J. Szpunar, Analyst 130, 442 (2005).
3. J. L. Gomez-Ariza et al., Anal. Chim. Acta 525, 15 (2004).