PARENT SESSION
1A Chemical, biological, and combined methods for the detection of pollutants
9:00 AM to 7:00 PM, Wednesday, 09 May 2001

(W/EH020) HPLC - ICP TOF MS for metal speciation on metallothioneins in carp cytosols.

Van Campenhout, Karen¹, Goenaga Infante, Heidi², Tian, Xiaodan ², Adams, Freddy ², Blust, Ronny ¹, ^{1 2}

ABSTRACT- Metallothioneins (MTs) are a group of low molecular non-enzymatic cysteine-rich-metal-binding proteins, which are resistant to thermocoagulation and acid precipitation. The interest in the determination and metal speciation of the different isoforms is based on their role in metal homeostasis, and detoxification processes of heavy metals. In consequence, there is a growing awareness of the need to determine the various MT isoforms and their metal binding characteristics rather than just the total MT concentrations. The classical techniques (ELISA, RIA, metal-saturation assays, polarography) are able to quantify the total MT concentrations, but fail to quantify separately the different MT isoforms or to provide information on their original metal composition. Selectivity in terms of isoforms and metal speciation can be achieved by the coupling of a high-resolution separation technique with an element selective detection technique. The coupling of High Performance Liquid Chromatography (HPLC) with Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has become a most popular hybrid technique for speciation of metal-binding proteins. Based on the advantages afforded by the use of ICP TOF MS (inductively coupled plasma time-of-flight mass spectrometry) as multi-element, multi-isotopic and fast detector for transient signal analysis, we have exploited the coupling of HPLC-ICP TOF MS for the characterization of metalloproteins in cytosolic fractions of different tissues (liver, kidney and gills) of carp exposed to Cd. The cytosolic proteins were separated on a calibrated gel filtration column using 30 mmol l^-1 Tris-HCl as mobile phase and detected by ICP TOF MS. The effect of Cd exposure on the profile of the cytosolic metal-binding proteins and their metal speciation was studied.

Key words: metallothionein, metal speciation, HPLC, ICP TOF MS