H. Chassaigne et R. Lobinski, POLYMORPHISM AND IDENTIFICATION OF METALLOTHIONEIN ISOFORMS BY REVERSED-PHASE HPLC WITH ONLINE ION-SPRAY MASS-SPECTROMETRIC DETECTION, Analytical chemistry (Washington), 70(13), 1998, pp. 2536-2543
Microbore reversed-phase HPLC with on-line ion-spray mass spectrometri
c detection is proposed for a study of polymorphism of rabbit liver me
tallothionein (MTRL) and its major isoforms MT-1 and MT-2. Separation
conditions had been optimized until each chromatographic peak could be
attributed to a single metalloprotein species, of which the molecular
mass could be determined by ion-spray MS. At the optimized conditions
(elution with the gradient 5-8% of acetonitrile within 50 min using a
5 mM acetate buffer at pH 6), the chromatogram of MTRL showed five pe
aks, whereas those of MT-1 and MT-2 showed nine and seven different pe
aks, respectively. The on-line determination of the molecular masses (
+/-1 Da) of the compounds eluted permits the unambiguous cataloguing a
nd further referencing of putative and true MT subisoforms. The result
s obtained are compared with those obtained by direct ion-spray MS of
the MT preparations at different pHs, with a goal to identify possible
chromatographic artifacts. Metals (Cd, Cu) in the eluted complexes we
re studied by HPLC with on-line ICP MS detection.