Speciation of Metallothionein-like Proteins of the Mussel Mytilus edulis at Basal Levels by Chromatographic Separations Coupled to Quadrupole and Double-Focusing Magnetic Sector ICPMS

Abstract

Characterization and partial purification of metallothionein-like proteins (MLPs) of the mussel Mytilus edulis from natural populations of three coastal regions in Spain were performed. Size exclusion chromatography (SEC) with quadrupole (Q-ICPMS) or double-focusing inductively coupled plasma mass spectrometry (DF-ICPMS) detection was used first for speciation of cadmium in such natural samples and those of mussels exposed to 500 mg·L^-1 Cd in an aquarium tank. SEC results showed always a single Cd−MLP peak (MLP fraction). The contents in Cd, Cu, and Zn of this MLP fraction, of the high molecular weight protein pool (HMW), and of the whole cytosol were then measured by DF-ICPMS. Then, a given aliquot (50 μL) of MLPs with the highest values for UV molecular absorption at 254 nm (also the maximum sulfur and Cd, Cu, or Zn contents) was used to further fractionation. Fast protein liquid chromatography “on line” with Q-ICPMS was used for the purpose. Two Cd-MLP isoforms (MLP-1, MLP-2), with retention times (t_R) of 15.7 and 16.0 min, were then detected in cytosols of the mussel samples of aquarium tank and also of the industrial area and Galicia coast. Conversely, wild coast mussels did not show any Cd-MLP signals at all. Analysis of essential elements copper and zinc in such cytosols by FPLC−Q-ICPMS revealed that these two metals were associated just to MLP-1. These results tend to indicate a different role for the two MLP isoforms detected in mussels (i.e., essential metals' homeostasis role seems to be tied to the MLP-1 isoform only). They illustrate the fact that trace metal speciation of unknown species in biological materials is becoming a challenge and points to the use of several complementary analytical techniques to obtain the required speciation information.