Abstract

The analytical potential of complementary chromatographic separations such as size exclusion (SEC) plus anion-exchange chromatography coupled with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS), was evaluated for the multi-elemental speciation of trace elements associated with methallothionein-like proteins (MLPs) in mussel hepatopancreas. The initial separation of the proteins is performed by "off-line" SEC, collecting fractions which are then analysed for the trace elements by ICP-TOF-MS in small sample volumes. Further purification is accomplished by the separation of the different proteins contained in the above obtained MLP fraction on a Mono Q (HR 5/5) anion-exchange column [fast protein liquid chromatography (FPLC)], using an ammonium acetate gradient at a physiological pH of 7.4 as the mobile phase. The separated proteins are detected "on-line" by UV absorption at 254 and 280 nm. Specific detection (16 metals) with on-line ICP-TOF-MS was also performed. This hybrid approach was applied to investigate the metal binding patterns of the proteins expressed by mussels which had been previously exposed to 16 trace metals in an aquarium tank for 1 and 2 weeks. The exceptional characteristics of ICP-TOF-MS for quasi-simultaneous monitoring of many trace elements in transient signals are illustrated by this "on-line" coupling to FPLC separation, paving the way to virtually simultaneous multi-elemental speciation of trace metals in biological material.