Characterization and quantification of metallothionein isoforms by capillary electrophoresis–inductively coupled plasma–isotope-dilution mass spectrometry

Abstract

In a new approach to the characterization and quantification of metallothionein isoforms an on-line isotope-dilution method in combination with the coupling of capillary electrophoresis (CE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) is reported. Metallothionein (MT) isoforms are separated by CE and the elements Cu, Zn, Cd, and S are detected simultaneously by use of ICP-SFMS in the medium resolution mode. On-line isotope dilution is performed by continuous introduction of an isotopically enriched, species-unspecific spike solution after the separation step. MT from rabbit liver and a further purified MT-1 isoform were quantified by determination of sulfur, and the stoichiometric compositions of the metalloprotein complexes are characterized by determination of their sulfur-to-metal ratios.

Inductively coupled plasma- and glow discharge plasma-sector field mass spectrometry : Part II. Applications

“…336,337 A reverse isotope dilution principle was used by Schauml€ offel et al in combination with capillary electrophoresis (CE) coupled to ICP-SFMS for metallothionein quantification: The sheath liquid supplied to the CE nebuliser interface was spiked with isotopically enriched metals and isotopically enriched sulfur ( 32 S or 34 S). 338 Due to the spectral interferences affecting all sulfur isotopes, the instrument was operated at medium mass resolution (3000), also for detection of the metals. In this way, possible interferences from 16 O 2 + or 16 O 18 O + , for example, were eliminated.…”

Online isotope dilution and inductively coupled plasma mass spectrometry: from elemental to species quantification

“…63,64 The application of online ID to quantify metallothionein (MT) isoforms presented good resolution when separated by HPLC coupled to ICP with ICP-TOFMS and ICP-QMS. The spike mass flow has been calibrated by reverse ID for online quantification of species separated by CE coupled to ICP-SFMS using separations at medium resolution.…”

“…The spike mass flow has been calibrated by reverse ID for online quantification of species separated by CE coupled to ICP-SFMS using separations at medium resolution. 63,64 In both works the determination of Cd, Cu, Zn and S in metallothionein isoforms allowed assessing the stoichiometric composition. The spike mass flow calibration of species separated by CE is laborious once calibration standards with eletrophoretic mobility are required.…”

Inductively Coupled Plasma–Mass Spectrometry (ICP-MS) for Quantitative Analysis in Environmental and Life Sciences: A Review of Challenges, Solutions, and Trends

“…108,[180][181][182] Based on this development, Prange and Schaumlöffel et al pioneered the field of sulfur isotopedilution analysis by combining postcolumn non-species-specific isotope dilution and capillary electrophoresis hyphenated to sector field ICP-MS for the highly resolved separation of different metallothionein (MT) isoforms, which allowed determination of the metal stoichiometry of the different isoforms as well as their absolute quantification. 183,184 A non-species-specific element spike containing 34 S, 65 Cu, 68 Zn, and 116 Cd introduced via the make-up flow of the CE interface was used for on-line isotope ratio measurement of 32 S/ 34 S, 63 Cu/ 65 Cu, 64 Zn/ 68 Zn, and 114 Cd/ 116 Cd. Reversed isotope dilution, using a standard solution containing S, Cu, Zn, and Cd with natural isotopic abundances, has been used for mass flow calibration of the different elements.…”

“…Based on the known MT-tosulfur stoichiometry, molar ratios between sulfur and the different metals, as well as the absolute amounts of the different isoforms were quantified. 183,185 This initial study has been further improved by Wang and Prange by using surface-modified CE capillaries to obtain a better separation of the different MT isoforms, which can be found in the commercially available preparations. Zn/protein ratio for the Aeromonas hydrophila (AE036) metallo-beta-lactamase.…”