Speciation of metallothionein-like proteins of the mussel Mytilus edulis by orthogonal separation mechanisms with inductively coupled plasma-mass spectrometry detection: effect of selenium administration

Several complementary separation mechanisms (size-exclusion chromatography, SEC; fast protein liquid chromatography, FPLC; reverse-phase chromatography, RPC) have been coupled to inductively coupled plasma mass spectrometry (ICP-MS) detection to investigate the speciation of Cd, Se, Cu and Zn in mussel hepatopancreas. SEC with double-focusing (DF) ICP-MS detection was used first for speciation analysis of those four trace elements, both in uncontaminated mussels and in mussels exposed to Cd (500 μg l −1) or to Cd+Se (500 μg l −1 of each element). Observed SEC results indicated that Se does not seem to significantly bind to metallothionein-like proteins (MLPs) ‘in vivo’. Total cytosolic Cd and MLPs content were lower in Cd+Se exposed mussels than those exposed to Cd only. For each treatment, 50 μl of the SEC peak fraction containing the MLPs was used to perform fast protein liquid chromatography coupled ‘on-line’ with a quadrupole (Q) ICP-MS. 82Se and 114Cd isotopes were simultaneously monitored. Four and five Cd/MLPs isoforms were then detected in mussels exposed to only Cd and Cd+Se, respectively. In contrast, no signs of Se/MLP isoforms were found for both treatments. Subsequently, the bulk of MLPs eluting from the FPLC system were isolated and lyophilizated. A 50-μl aliquot of such reconstituted lyophilisate was then injected into a Vydac C 8 Reverse-Phase column directly connected to the Q-ICP-MS. Results confirmed the presence of one more Cd/MLP peak in those mussels exposed to Cd+Se. However, the number of Cd/MLP peaks detected decreased to three and four in only Cd and Cd+Se exposed animals, respectively. These results tend to indicate that Se, which does not trigger the biosynthesis of MLPs, could probably orient such synthesis towards the generation of a new Cd/MLP isoform in mussels submitted to both elements. The possible interrelation/complementation between Se and MLP against Cd toxicity is discussed.