Identification of a Metallothionein in Synechococcus by Capillary Electrophoresis Hyphenated with Inductively Coupled Plasma Mass Spectrometry

Abstract

In 1957, Margoshes and Vallée 1 discovered a new type of protein, which seemed to contain metal ions in its structure. However, the first paper confirming the presence of cadmium in an isolated protein, named metallothionein (MT), was only published in 1960 by Kagi and Vallée. 2 Metallothioneins are proteins with low molecular mass, rich in cysteine residues and extremely reactive to bivalent metals. 3 These proteins are responsible for processes related to both intracellular absorption and the regulation of biologically essential elements, such as zinc and copper. MTs are also responsible for cell protection from toxic elements, such as cadmium and mercury. 4,5 Metallothioneins are present in mammals and also in eukaryote and prokaryote organisms. 3,6 In the majority of animal species, MTs are abundant in the liver, pancreas, intestines and kidney tissues. The concentration of these proteins depends on both the species and the tissues, and reveals the diet effect in each organism. 7 Metallothioneins are usually determined by employing classical methodologies, such as metal-ligand assays with mercury 8 and cadmium. 9 Among those classical methodologies are also found electrochemical techniques based on the quantification of MT fractions via the oxidation of sulfhydryl groups, 10 and also immunology techniques, such as radioimmunoassay 11 (RIA), and enzymatic assays in sorbents 12 (ELISA). However, methods involving metal saturation or electrochemical techniques undertake interferences due to oxyreduction reactions of other species present in the samples, 12 what makes them inefficient to quantify the metal concentration in the isoforms. 13 This drawback was overcome when anionexchange high-performance liquid chromatography (HPLC) was employed in the separation of the two main MT isoforms. 14 Subsequently, reverse-phase HPLC was also employed to improve the resolution in the separation of isoforms.15,16 HPLC was coupled to atomic absorption spectrometry (AAS) by using a T-shaped interface to determine Cd, Zn and Cu in MT from rabbit liver in µg g -1 level. 17,18 Other couplings, such as RP-HPLC with inductively coupled plasma mass spectrometry 19, and RP-HPLC with electrospray mass spectrometry 21,22 (ES-MS), were also proposed for the separation and determination of Cd and Zn in MT. Nevertheless, certain demands related to the utilization of organic solvents and column specificities restrict the use of RP-HPLC as a separation method. 23 In the last decade, capillary-zone electrophoresis has been successfully employed in the separation of proteins and peptides, especially for those with low molecular mass. 24,25 Despite its high efficiency, capillary electrophoresis (CE) presents some limitations related to the low sensitivity of UV detectors, which are widely employed in CE. 26 In this sense, it is not unexpected that several efforts have been made to couple CE with ICP-MS in order to overcome sensitivity limitations. However, there are some practical aspects, such as capillary positioning in the interface, flo...

Molecular screening of microbial communities for candidate indicators of multiple metal impacts in marine sediments from northern Australia

“…Taxa that were identified as sensitive to multiple metals included most Crenarchaeota and Methanomicrobia (Archaea); and Cyanobacteria, Chloroflexi, Pseudoalteromonadaceae, and Verrucomicrobiales DEV007 (Bacteria). However, some of these sensitive taxa (particularly Cyanobacteria) have been previously well described as metal tolerant , and the metal susceptibility profiles described in the present study may have been influenced by confounding factors, as discussed below in the section Confounding factors . Because of the complexity of these sediments, and the potential for confounding factors that may bias analysis, culture and resistance testing of organisms identified in this screening process are required to confirm metal resistance profiles.…”

Analytical Methods for Metallothionein Detection

“…The advantage of this type of detection is the relative versatility in the deep UV range; on the other hand this is the main reason of interferences. Therefore other detection types such as CE with inductively coupled plasma mass spectrometry (ICP-MS) are tested [130,131]. The main limitation of this method has been the coupling of the separation and ionization.…”

Capillary electrophoresis of proteins 2003–2005

“…coccus [248], conformational changes in HSA [249], metal-binding proteins [250], ricine and its subunits [251], capsid proteins from human rhinovirus [252,253], antithrombin III from human plasma [254], prostate-specific antigen isoforms [255], human fibrinopeptides [256], ribonuclease H [257], endogenous extracellular signalregulated protein kinase [258], collagen methylation [259], half antibody in IgG(4) [260], and detect bone morphogenic protein-2 in rat vascular smooth muscle cells [261,262].…”