Rabbit liver metallothionein subisoform characterisation using liquid chromatography hyphenated to diode array detection and electrospray ionisation mass spectrometry

The direct electrospray ionisation mass spectrometry infusion of rabbit liver MT and of its two isoforms in an acidic medium has allowed the identification of the already sequenced (apo) subisoforms MT-1a, MT-2a, 2b, 2c, 2d and 2e, but also shows the presence of minor quantities of non-acetylated subisoforms and that of unsequenced forms. Using liquid chromatography hyphenated to diode array detection and electrospray ionisation mass spectrometry, the nonacetylated apoforms have been shown to have different retention times than their corresponding acetylated forms. In many cases, a single molecular mass and therefore a single compound can be attributed to each chromatographic peak (case of aposubisoforms), but this is not always the case as some HPLC peaks obviously contain several species which are very often different complexes of the same subisoform at a particular pH. Interestingly, the results show clearly that the non-acetylated forms are the first species which start to be complexed, first by Cu and then by Cd, when the pH increases from 1.7 to 5 and this holds true for all subisoforms. For their part, the major acetylated subisoforms such as MT-1a, MT-2a or MT-2e starts to be significantly complexed at higher pH values (around pH 4).