Detection of artefacts and peak identification in reversed-phase HPLC of metallothioneins by electrospray mass spectrometry

The use of ion-spray mass spectrometry rendered it possible to characterize the signals obtained during studies of the polymorphism of metallothionein (MT) by reversed-phase (RP) HPLC in terms of the molecular mass. Artefact signals due to incomplete metallation, exchange of metals with the impurities of the column stationary phase and cross-contamination of the preparations purified by size-exclusion and anion-exchange chromatography may be present. On the other hand, some signals in RP HPLC with UV detection considered to belong to a single species were found to be composed of several complexes eluting precisely at the same time. On-line electrospray mass spectrometry was used to systematize the knowledge of the MT isoforms and subisoforms by attributing to each of the eluting peaks the molecular mass of the form involved and can be used to compare the results obtained for the different groups.