Selenomethionine analysis

Membrane-integrated proteins were always hard to express in cell-based systems in sufficient quantity for structural analysis. In cell-free systems, they can be produced on a milligrams per milliliter scale, which, combined with labeling with stable isotopes, is also very amenable forNMR spectroscopy [157-161]. Possible applications of in vitro expression systems also include incorporation of selenomethionine (Se-Met) into proteins for multiwavelength anomalous diffraction phasing of protein crystal structures [162], Se-Met-containing proteins are usually toxic for cellular systems [163]. Consequently, rational design of more efficient biocatalysts is facilitated by quick access to structural information about the enzyme. 

Figure 8.4. Cation exchange HPLC-ICP-MS analysis of 77Se-enriched yeast using two enzymatic sample preparation strategies (a) sequential use of /3-glucosidase and a protease mixture (b) protease XIV and (c) mass balance of selenium and selenium species fractionated during the sample preparation and HPLC separation procedure. SeMet = selenomethionine SeOMet = selenomethionine Se-oxide. Reproduced from [133] by permission of The Royal Society of Chemistry.

Se Speciation in Plants The presence of a number of volatile Se species was reported in edible allium plants such as garlic by GC-AES [79]. Selenomethionine, Se-methylselenocysteine, and y-glutamyl-Se-methyl-L-seleno cysteine were identibed in garlic and onion by HPLC-ICP-MS and ES-MS/MS [80]. Selenomethionine is the primary species found in all types of nuts (19D25 percent of the total Se) [30], sunBower [81], and mushrooms [36, 37], The distribution of Se among different fractions (lipid extract, low molecular weight, and protein fractions) of nuts and speciation analysis was studied [30]. Selenium was not detected in any of the lipid extracts obtained from the different types of nuts [30], Results obtained for Brazil nuts by SEC with on-line ICP-MS detection showed that approximately 12 percent of total Se was weakly bound to proteins [30],... 

Selenium Speciation in Edible Animal Tissues Reports on Se specia-tion analysis in edible animal tissues have been scarce. Speciation analysis of Se in cod muscle tissue was performed by separating the species using both RP- and SE-HPLC prior to ICP-MS detection. The main Se compound found in enzymatic hydrolysates was selenomethionine [42], This selenocompound was absent in MeOHDHCl extracts, indicating that Se was mainly incorporated into proteins. A number of unidentiFed Se species were also detected in cod muscle tissue, the separated Se compounds being quantised on-line by post-column isotope dilution [42], Soluble Se compounds extracted from muscles of chicken, turkey, duck, ostrich, lamb, cattle, and pig were separated by SEC with ICP-MS detection. Four peaks were observed, but distribution of Se among these peaks varied considerably in tissues from different animal species [86]. 

L. Hinojosa Reyes, F. Moreno Sanz, P. Herrero Espilez, J. M. Marchante-Gayon, J. I. Garcia Alonso, A. Sanz-Medel, Biosynthesis of isotopically enriched selenomethionine application to its accurate determination in selenium-enriched yeast by isotope dilution analysis-HPLC-ICP-MS, J. Anal. Atom. Spectrom., 19 (2004), 1230-1235. 

Enzymes such as protease in conjunction with pancreatin and amylase have been extensively used to liberate Se species from proteins for analysis [43, 57, 128, 133-136]. Relatively long times ( 24 h) are required to fully hydrolyze proteins using enzymes. However, not all Se is released as simple amino acids. Some peptides, and small molecular weight proteins remain. Thus, ultrafiltration (< 1 kDa) before analysis will be needed to separate amino acids from other material with higher molecular weight. In the presence of cysteine, selenomethionine and selenocysteine are stable to enzyme attack (Fig. 20.2). However, although large amounts of Se are released from marine tissues (30-60 percent), little (less than 10-20 percent) is characterizable by HPLC-ICP-MS. 

Enhancing the Se levels in crops can be achieved by adding organic amendments (manure of Se-supplemented farmed animals) or inorganic Se to mineral fertilizers [116, 117]. The use of sodium selenate-enriched fertilizers in Finland resulted in increased Se levels in different foods and, consequently, the average serum Se in the population improved over the period 1984 D1988. ICP-MS was used to study the feasibility of wheat enrichment by selenate addition to soil fertilizers [118]. AE-HPLC-ICP-MS was optimized for the separation of selenite, selenate, selenocysteine, and selenomethionine. Total Se determination and speciation analysis were performed in water extracts and in enzymatic digests of wheat samples. It was shown that a major part of the selenate taken up by cereals was converted to selenomethionine. 

Since selenized yeast is used in human nutrition, there is a need for full characterization of Se species. However, even more importantly, the quantitative analysis of biologically active selenomethionine is demanded. After a long-term intake of selenized yeast, the undesired accumulation in proteins occurs with increased... 

C. Devos, K. Sandra, P. Sandra, Capillary gas chromatography inductively coupled plasma mass spectrometry (CGC-ICPMS) for the enantiomeric analysis of d,l-selenomethionine in food supplements and urine, J. Pharm. Biomed. Anal, 27 (2002), 507D514. 

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. ... 

The principle of the isotope dilution analysis (IDA) is described in Section 6.4. Due to its advantages as a definitive and accurate analytical method for the determination of element concentration via isotope ratio measurements, IDA is being increasingly applied in mass spectrometry, especially in ICP-MS and LA-ICP-MS as one of the most frequently used techniques. For example, the isotope dilution technique is employed in species analysis in biological systems, " e.g., for the determination of mercury species in tuna material,or in aquatic systems. Further applications of the isotope dilution technique are the determination of selenomethionine in human blood serum by capillary HPLC-ICP (ORC) MS ° or sulfur speciation in gas oil, diesel or heating fuel by LA-ICP-MS. Evans and co-workers have reported on the high accuracy analysis of sulfur in diesel fuel by IDA. ICP-SFMS has been employed for Si species analysis in biological or clinical samples and... 

Isotope ratio measurements were employed for the quantification of analytical data using the isotope dilution strategy. For example, isotope dilution analysis was developed by Sanz-Medel s group for the determination of selenomethionine in Se enriched yeast material by HPLC-ICP-MS using a Se-enriched selenomethionine spike obtained by growing yeast on a Se rich culture medium. For Cr(III)/Cr(VI) determination in yeast, Caruso et al employed the double spike species specific isotope dilution technique measured by HPLC-ICP-MS. The isotope pattern deconvolution approach applied in this work delivers a more intuitive and elegant solution to an otherwise complex data analysis without the need for iterative calculations as widely practised in double spike isotope dilution. The results are in exact agreement with the conventional isotope dilution calculations. ... 

Egerer-Sieber, C., Herl, V., Miiller-Uri, R, Kreis, W. and Muller, Y.A. (2006) Crystallization and preliminary crystallographic analysis of selenomethionine-labelled progesterone 5p-reductase from Digitalis lanata Ehrh. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 62, 186-8. 

Hendrickson, W. A., Horton, J. R., and LeMaster, D. M. (1990). Selenomethionine proteins produced for analysis by multiwavelength anomalous diffraction (MAD) a vehicle for direct determination of three-dimensional structure, EMBO J., 9, 1665-1672,. 

NMR utihzes the magnetic properties of number of nuclei [4,5], However, from the point of view of peptides analysis only few isotopes can be though as a structural probes. The most important are H, and N isotopes representing the basic building units of peptides. In some cases, Se or nuclei are employed in structural analysis when appropriate derivatives (cysteine, selenomethionine or phosphorylated peptides) are under investigation. AH of the above mentioned nuclei have 14 spin and are relatively easy to measure however, in many cases simple one-pulse acquisition fails. Below, we will show how today these problems can be overcome. 

Encinar et al. developed a method for the accurate determination of sele-noamino acids in human serum by species-specific isotope dilution analysis. A human serum was enzymatically digested, and then the selenoamino acid and carboxymethylated selenocysteine were separated and quantified by HPLC-ICP-MS. Quantification of selenomethionine was carried out by isotope dilution using a synthetic Se-labeled counterpart. The selenomethionine in samples was also measured by using selenomethionine as an internal standard. The instrumental detection limit was down to 75 fg Se and the precision was better than 5% RSD. ... 

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