Comparison of capillary electrophoretic techniques for analysis and characterization of metallothioneins

Abstract

To explore and understand the significance of individual metallothionein isoforms, the methods of their identification are needed. Separation of these isoforms requires a high resolution technique which can exploit very small differences in mass, charge, and hydrophobicity. In this report, three different techniques of CE were analyzed and used for metallothionein separation: detection using capillary gel electrophoresis, capillary zone electrophoresis, and capillary isoelectric focusing. Also, three different metallothionein samples were used from horse kidney, rabbit liver, and human liver. We identified metallothionein isoforms based on the determination of their relative molecular masses, on the charge differences in different pH buffers, and based on the pI value. Methods used in this report allow metallothionein identification, permit to quantify the purity and content of its isoforms, and allow studying its polymerization. This report supports and endorses the increased application of CE methodology in proteomics.

Capillary electrophoretic separation of serum proteins of workers occupationally exposed to heavy metals

“…CE is a liquid-based system that uses small intradiameter (10-100 µm) bare fused-silica capillaries. CE enables separation of protein isoforms [19], the identification of protein modifications [20] and quantitative and qualitative analyses of changes in serum protein profiles [21]. CE can be used as a screening test of diseases connected with changes in serum proteome [22].…”

The application of capillary electrophoresis, mass spectrometry and Brdicka reaction in human and rabbit metallothioneins analysis

“…Capillary electrophoresis is often used to analyze and separate MT isoforms from different organisms and tissues. 11,23 It can also be used for monitoring MT concentration in biological samples. 23 Křížková et al were the first to propose a chip CE technique for the analysis of MT, i.e., of the effect of oxidation on the structure of MT and its ability to form aggregates and polymers.…”

“…Human and rabbit MTs have previously been analyzed by western blot, ELISA, capillary electrophoresis (CE), and high-performance liquid chromatography. [10][11][12][13][14][15] Therefore, the purpose of the research was the comprehensive characterization of human and rabbit MTs. Modern electrochemical and advanced analytical approaches such as the Brdicka reaction, chip CE and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to characterize and compare human and rabbit MTs.…”

Element Speciation Analysis Using Capillary Electrophoresis: Twenty Years of Development and Applications

“…These small cysteine-rich metalloproteins exist naturally as a mixture of up to four major isoforms with varying Cd, Zn, and Cu content and multifunctional physiological behavior. Since they present complex metal-speciation patterns in mammals, varying from one animal type to another, MTs force CE to operate with the maximum attainable resolution rather than the minimum required. Even in individual commercial preparations, there could appear to be three or four peaks, for example, in the Cd-specific electropherograms …”