[Characterization method for metallothioneins by reversed-phase liquid chromatography coupled with electrospray...

Mammalian metallothioneins (MTs) usually have two major isoforms, MT-1 and MT-2, existing in tissues because of the genetic polymorphism. Metallothionein isoforms and sub-isoforms were characterized with a hyphenated technique of reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The MT-isoform separation was carried out using a C8 narrow-bore column Vydac C8 (250 mm x 2.1 mm i.d., 5 microm, 30 nm). Buffer A was 5 mmol/L ammonium acetate in water (pH 6.0) and buffer B was 5 mmol/L ammonium acetate in methanol-water (1:1, v/v) (pH 6.0). A linear gradient elution was used by increasing buffer B from 10% to 37.5% within 40 min. The flow rate was set at 0.20 mL/min. The tentative formulas of MT isoforms and subisoforms were interpreted with the measured masses at the vicinity of apexes in total ion current chromatogram of ESI-MS and the reference data reported previously. The method was validated with the standard reference materials of MT-1 and MT-2. The results indicated that many MT species interpreted were in good agreement with the reference data reported. The MT sample from rabbit liver was measured with the established method. The results, of which the apo-MT was used by acidification of the sample for simplifying the mass spectra, showed that there were species of MT isoforms and subisoforms in rabbit livers.