Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS

Abstract

Pneumatically assisted electrospray mass spectrometry (ESMS) in the direct mode and as a chromatographic detection technique was developed for the characterization of horse kidney metallothionein isoforms. Direct analysis in an acidic medium showed the presence of three major and five minor isoforms, the molecular masses of which were determined. The presence of the major isoforms (two of which matched the molecular masses calculated according to the published sequences) was confirmed by complexation with Cd at pH 4.0 and the determination of the stoichiometry of the complexes formed. Reversed-phase chromatography of Cd₇–MT complexes (pH 6.0) gave two signals corresponding to the MT-1A and MT-1B isoforms. A post-column acidification procedure was developed to eliminate the possibility of artefacts associated with the formation of mixed-metal (Cd, Zn) complexes during chromatography in neutral media, and to improve the accuracy of the determination of the molecular mass of MT isoforms.