RESUMEN
An on-line capillary electrophoresis-mass spectrometry method (CE-MS) for the detection of metallothionein (MT) isoforms is described. The detected masses were usually within 1-1.5 mass units of the expected molecular weights. MT-containing samples from rabbit, sheep, and yeast (Saccharomyces cerevisiae) were subjected to CE-MS analysis. The analysis of rabbit liver MT revealed the masses of 10 proteins/peptides. Five of the detected masses corresponded well with the expected masses calculated from the amino acid sequence of previously described MT isoforms, one was suspected to be a deacetylated form of MT-2A, one was presumed to be a yet unknown isoform, and three masses were classified as non-MT compounds. From the analysis of a fetal sheep liver extract six proteins were detected of which three masses corresponded to previously described MT isoforms. Two purified MT subforms from S. cerivisiae (encoded by the CUP1 locus) were analyzed for their copper content and both forms were found to contain eight copper atoms per molecule.
Asunto(s)
Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Metalotioneína/análisis , Saccharomyces cerevisiae/química , Animales , Cobre/química , Isomerismo , Hígado/química , Hígado/embriología , Peso Molecular , Conejos , Ovinos , Especificidad de la EspecieRESUMEN
A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase preconcentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)-CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was < 10% and usually < 5% while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 microgram/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50% increasing to almost 70% by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE-CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms.