Characterization of metal complexes with metallothioneins in the liver of the carp Cyprinus carpio by reversed-phase, HPLC with ICP-MS and electrospray ionization (ESI)-MS

Inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) and electrospray ionization mass spectrometry (ESI-MS) were used in parallel in combination with two-dimensional chromatography (size-exclusion followed by reversed-phase HPLC) for the characterization of metal complexes with metallothionein (MT) isoforms in hepatic cytosols of Cd exposed carp. The cytosolic MT fraction was partially purified by size-exclusion (SE) HPLC, preconcentrated and de-salted by centrifugal microfiltration. The separation of the metal complexes with MT isoforms was performed by reversed-phase (RP) HPLC using a gradient up to 30% methanol in acetate buffer (pH 7.4). Two major and several minor chromatographic peaks were distinguished by RP-HPLC-ICP-TOFMS within a chromatographic run of 20 min. Applying ESI-MS as a detector for RP HPLC allowed the identification of the two major signals detected by ICP-MS on the basis of the molecular mass determined on-line for the metallocomplex(es) and for the ligand. Mass spectra taken at the peak apices indicated the co-elution of different metal (Cd, Cu, Zn and Pb) complexes with MT isoforms within each peak. A simple in-source protein oxidation procedure was used to obtain the ligand of the complexes (oxidized form) on arrival at the detector; direct information was obtained on the molecular mass of two MT isoforms in carp liver, allowing their identification. The efficiency and robustness of this method were demonstrated using a rabbit liver MT1 standard as test sample.