Polymorphism and identification of metallothionein isoforms by reversed-phase HPLC with on-line ion-spray mass spectrometric detection.
Microbore reversed-phase HPLC with on-line ion-spray mass spectrometric detection is proposed for a study of polymorphism of rabbit liver metallothionein (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 ionspray 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 peaks, whereas those of MT-1 and MT-2 showed nine and seven different peaks, respectively. The on-line determination of the molecular masses (+/- 1 Da) of the compounds eluted permits the unambiguous cataloguing and further referencing of putative and true MT subisoforms. The results 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 were studied by HPLC with on-line ICP MS detection.
Chassaigne
H
H
Laboratoire de Chimie Bio-inorganique et Environnement, CNRS EP132, Pau, France.
Lobinski
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003300:Copper / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry; D008668:Metallothionein / Q000032:analysis* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011110:Polymorphism, Genetic; D011817:Rabbits; D013237:Stereoisomerism
D002104:Cadmium; D003300:Copper; D008668:Metallothionein