Speciation metallothioneins

SEC has been used extensively to separate proteins containing different elements such as Zn (II), Cd (II), Cu (H), and Hg (II) in metallothionein [78-80]. These studies show the potential of SEC-ICP-MS for metalloprotein separations but are not specifically concerned with elemental speciation. 

In past years, on line chromatographic coupling techniques such as HPLC and CE coupled to ICP-MS with the isotope dilution technique have been used for element quantification in speciation analysis. An interesting application of the isotope dilution technique in medical research was proposed recently by Prange and co-workers, who added highly enriched " S, Cu, Zn and Cd spikes to the interface of the CE-ICP-MS system. The authors separated isoforms of metallothionein (e.g., of rabbit liver) by capillary electrophoresis and quantified S, Cd, Cu and Zn concentrations in isoforms by ICP-SFMS using the isotope dilution technique. A new selenized yeast reference material (SELM-1) for methionine, selenomethionine (SeMet) and total selenium content has also certified by an intercomparison exercise. ... 

Element-specific detection by ICP-MS has been widely used in the characterization of metallothioneins (MTs). The biological importance of these proteins is due to their role in homeostatic regulation of essential heavy metals like Cu and Zn. On the other hand, MT protects the cells from harmful chemicals, like nonessential and excessive essential heavy metals, reactive oxygen species, radicals, and alkylating agents. Fararello et reviewed different chromatographic approaches with ICP-MS detection for the multielemental speciation in MTs and MT-like proteins. 

Richarz, A.-N., Bratter, P. Speciation analysis of trace elements in the brain of individuals with Alzheimer s disease with special emphasis on metallothioneins. Anal. Bioanal. Chem. 372, 412-417 (2002)... 

Suzuki, K.T. Hyphenated techniques as tools for speciating biological metals Metallothionein and metal-binding proteins. Analusis 1998, 26 (6), M57-M61. 

The most definitive assessment of the metal composition of metalloproteins comes from the application of element-specific detection methods. CE-ICP-MS provides information not only about the type and quantity of individual metals bound to the proteins but also about the isotopes of each element as well [11,12]. Elemental speciation has become increasingly important to the areas of toxicology and environmental chemistry. Such analytical capability also opens up important possibilities for trace element metabolism studies. Figure 1 depicts the separation of rabbit liver metallothionein containing zinc, copper, and cadmium (the predominant metal) using CE-ICP-MS with a high-sensitivity, direct injection nebulizer (DIN) interface. UV detection (200 nm) was used to monitor the efficiency of the CE separation of the protein isoforms (MT-1 and MT-2), whereas ICP-MS detection made it possible to detect and quantify specific zinc, copper (not shown), and cadmium isotopes associated with the individual isoform peaks. 

Rgure 2 Electrophoretic separation of fish Cd-MTs complexes (real sample) by CE-ICP(Q)MS. Signals for " Cd and " Cd were monitored showing that Cd binds two main MT isoforms. (Alvarez-Uamas G, Fernandez de la Campa MR, and Sanz-Medel (2003) Sample stacking capillary electrophoresis with ICP-(Q)MS detection for Cd, Cu, and Zn speciation in fish liver metallothioneins. Journal of Analytical Atomic Spectrometry 18 460-466 reproduced from The Royal Society of Chemistry.)... 

Prange A and Schaumloffel D (2002) Hyphenated techniques for the characterization and quantification of metallothionein isoforms. Analytical and Bioanalytical Chemistry 373 441 53. Sanz-Medel A, Montes-Bayon M, and Fernandez Sanchez ML (2003) Trace element speciation hy ICP-MS in large hiomolecules and its potential for proteomics. A review. Analytical and Bioanalytical Chemistry 377 236-247. Suzuki KT (1991) Metallobiochemistry Part B, metallothionein and related molecules. In Riordan JF and VaUee BL (eds.) Methods of Enzymology, vol. 205, pp. 252-262. San Diego Academic Press. 

Hgure 7 Multielement speciation of MTs by FPLC coupled online to ICP-MS and UV-visible spectrometry. (Reprinted from Ferrarello CN, Fernandez de la Campa MR, Carrasco JF, and Sanz-Medel A (2000) Speciation of metallothionein-like proteins from the mussel Mytilus edulis at basal levels by chromatographic separations coupled to quadrupole and double focussing magnetic sector. ICP-MS. Analytical Chemistry 72 5874-5880.)... 

Figure 8 New trends in speciation Separation of Cd-metallothionein isoforms by (A) SEC with UV and ICP-MS detection, (B) reversed-phase-LC-ICP-MS of the main Cd containing species eluted by SEC, (C) ES-MS of the peaks labeled as 1 and 2 in the RP-LC-ICP-MS chromatogram. (Reproduced from Polec K, Garcfa-Arribas O, Perez-Calvo M, etal. (2000) Identification of cadmium-bioinduced ligands in rat liver using parallel HPLC-ICP-MS and HPLC-electrospray MS. Journal of Analytical Atomic Spectrometry 15 1363-1368.)...

The ease of sample introduction by nebulization and the rapid multielement detection capabilities of ICP-MS lend the technique to online detection for metal and metalloprotein speciation by liquid chromatographic separations. This approach of LC/HPLC-ICP-MS has been applied to speciation of organo-Sn [50], organo-Pb [51], and organo-As [52], separation of metalloproteins such as metallothionein and ferritin [53,54], analysis of Au metabolites in blood from patients undergoing chrysotherapy for arthritis [55], and separation and quantitation of macromolecular forms of Fe in human liver biopsies [56]. 

Alvaeez-Liamas G., Fernandez De La Campa M. R., Fernandez Sanchez M. L. and Sanz-Medel A. (2002) Comparison of two CE-ICP-MS interfaces based on microflow nebulizers application to cadmium speciation in metallothioneins using quadrupole and double-focusing mass analyzers,... 

Ferrarelo, C. N., Encinar, Ruiz, J., Centineo, G., Alonso, J. I. Garcia, de la Campa, M. R. Fernandez, and Sanz-Medel, A. (2002) Comparison of three different ICP-MS instruments in the study of cadmium speciation in rabbit Uvct metallothionein-1 using tevCTsed-phase HPLC and post-column isotope dilution analysis. J. Anal. At. Spectrom., 17,1024. 

In species-unspecific isotope dilution techniques, an isotopically enriched standard solution is added post-column, and the data evaluation is performed as described in Section 7.3.4 for CE coupling. Species-unspecific isotope dilution for HPLC coupling has been described for the determination of Cd, Cu and Zn in metallothioneins extracted from eel liver, while Fe, Cu and Zn were determined in proteins separated from human serum. This technique has also been applied to iodine determination in humic substances,as well as selenium speciation in human serum. Species-specific isotope dilution using HPLC-ICP-MS has been described for selenium analysis, the analysis of organotin species in sediments, methylmercury in fish tissue, trimethyllead in water, and chromium speciation. Overviews to this topic have been published, e.g. by Heumann and Hill. ... 

Two papers have been published on the speciation of sulfur using ICP-DRC/CC-MS as a detector. ICP-MS with an octopole cohision cell was used for the detection of a CE separation of metallothionein-like proteins. The collision gas (Xe) and cell conditions were selected to reduce oxygen interference ( 02+) on the main sulfur isotope A completely different approach was taken by Hann et al. who introduced O2 as the reaction gas in the collision ceU, and measured the reaction product As obtaining metal-to-sulfur ratios in... 

Rodriguez-cea, A., Fernandez de la Campa, M.R., Blanco-Gonzdlez, E., Andon Ferndndez, B., and Sanz-Medel, A. (2003) Metal speciation analysis in eel Anguilla an-guilla) metallothioneins by anionic exchange-EPLC-isotope dUution-ICP-MS. 7. Anal At. Spectrom., 18,1357-64. 

Crews et al. have applied their size-exclusion separations to a study of Cd speciation in pig kidney. Cadmium in different proteins could be distinguished in cooked, uncooked, and digested pig kidney. The bulk of the soluble cadmium in retail pig kidney was associated with a metallothionein-like protein, some of which survived cooking and digestion. The detection limits of ICP-MS were sufficient to permit studies of Cd speciation at normal levels of Cd previous work on this subject by other methods was generally restricted to abnormally high levels of Cd [45]. 

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