Capability of flatbed electrophoresis (IEF and native PAGE) combined with sector field ICP-MS and autoradiography for the speciation of Cr, Ga, In, Pt and V in incubated serum samples

Abstract

The capability of flatbed electrophoresis combined with sector field ICP-MS (ICP-SFMS) and autoradiography is reported. Human serum and rabbit serum were separated by IEF and native PAGE using a PhastSystem unit (Amersham Pharmacia Biotech, Uppsala, Sweden). Rabbits (Flemish Giant) received intraperitoneal injections of ‘cold’ Ga(III) and In(III) in physiological buffer. Human serum was incubated in vitro with the radiotracers ⁵¹ Cr(III), ¹⁹¹ Pt(II) or carrier-free ⁴⁸ V(V). Measurement of the protein-bound metals followed two different strategies: (A) ICP-SFMS, in which each rabbit serum sample was run twofold and, after separation, one gel was silver stained and the other replicate was cut into segments, which were digested and measured for Ga and In by ICP-SFMS; (B) autoradiography, in which, after separation, the gels were exposed to a phosphorus screen at –20 °C, an autoradiogram was obtained by laser densitometry and subsequently the proteins were detected by silver staining. Both strategies have advantages and drawbacks, but both are powerful and highly sensitive and allow the attribution of the respective metals to serum proteins. The suitability of these methods for trace element speciation depends strongly on the stability of the trace element-protein binding.