Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry.
A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.
Prange
A
A
GKSS Forschungszentrum, Geesthacht, Germany. andreas.prange@gkss.de
Schaumlöffel
D
D
Brätter
P
P
Richarz
A N
AN
Wolf
C
C
D000368:Aged; D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D002850:Chromatography, Gel; D003600:Cytosol / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D013058:Mass Spectrometry; D008668:Metallothionein / Q000032:analysis*; D008670:Metals / Q000032:analysis; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000032:analysis
D008670:Metals; D013455:Sulfur; D008668:Metallothionein