Characterization of metallothionein isoforms by reversed-phase high-performance liquid chromatography with on-line post-column acidification and electrospray mass spectrometric detection

Post-column acidification of the chromatographic effluent was developed to eliminate artefacts in investigations of the polymorphism of metallothionein (MT) by microbore reversed-phase HPLC with detection by pneumatically assisted electrospray mass spectrometry. Metallated species (Cd, Zn and mixed Cd-Zn complexes) were decomposed on-line to produce apo metallothioneins of which the molecular masses were determined by MS. Besides the simplification of the mass spectra taken at the apexes of the chromatographic peaks, the method resulted in a 10-fold improvement of the detection limit of metallothionein and allowed a more comprehensive and less ambiguous detection and identification of the iso- and subisoforms, The method was applied to the characterization of rabbit liver metallothioneins: rabbit liver MT (purified by size-exclusion chromatography only) and MT-1 and MT-2 isoform fractions purified additionally by anion-exchange chromatography. (C) 1998 Elsevier Science B.V. All rights reserved.