A High-Throughput Absolute Quantification of Protein-Bound Sulfur Amino Acids from Model and Crop Plant Seeds.

In this procedure, we describe a high-throughput absolute quantification protocol for the protein-bound sulfur amino acids, cysteine (Cys) and methionine (Met), from plant seeds. This procedure consists of performic acid oxidation that transforms bound Cys into cysteic acid (CysA) and bound Met into methionine sulfone (MetS) followed by acid hydrolysis. The absolute quantification step is performed by multiple reaction monitoring tandem mass spectrometry (LC-MS/MS). The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common acid hydrolysis protocol, followed by water extraction that includes DL-Ser-d3 and L-Met-d3 as internal standards to enable the quantification of the absolute levels of the protein-bound Cys and Met with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples from Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound cysteine and methionine from seeds.

Methionine supplementation spares body protein by regulating the expression of mTORC1 downstream factors in rats fed a soy protein diet with sufficient sulfur amino acids: a pilot study.

Dietary supplementation with methionine and threonine spares body protein in rats fed a low protein diet, but the effect is not observed for other essential amino acids. Although the requirement for sulfur amino acids is relatively high in rodents, the precise mechanisms underlying protein retention are not fully understood. The aim of this study was to explore whether the activation of mammalian target of rapamycin complex 1 (mTORC1) downstream factors in skeletal muscle by supplementation with threonine and/or methionine contributes to protein retention under sufficient cystine requirement. Male Sprague-Dawley rats were freely fed a 0% protein diet for 2 weeks. These experimental rats were then fed a restricted diet (14.5 g/day) containing 12% soy protein supplemented with both cystine and, methionine and threonine (MT), methionine (M), threonine (T), or neither (NA) (n = 8) for an additional 12 days. Two additional groups were freely fed a diet containing 0% protein or 20% casein as controls (n = 6). Body weight and gastrocnemius muscle weight were higher, and blood urea nitrogen and urinary nitrogen excretion were lower, in the M and MT groups than in the T and NA groups, respectively. p70 S6 kinase 1 abundance was higher, and eukaryotic translation initiation factor 4E-binding protein 1 abundance and mRNA levels were lower, in the skeletal muscles of the M and MT groups. These results suggest that methionine regulates mTORC1 downstream factors in skeletal muscle, leading to spare body protein in rats fed a low protein diet meeting cystine requirements.

Difference in the nutritional, in vitro, and functional characteristics of protein and fat isolates of two Indian chia (Salvia hispanica L) seed genotypes with variation in seed coat color.

This study aimed to determine the association between the seed coat color of two chia seed genotypes for their composition, protein content, amino acid, and fatty acid profiles. The optimal pH for protein isolation for both genotypes (BCPI and WCPI) was 10, based on protein purity and solubility. Fatty acid profiling indicated, overall, 18 different fatty acids higher in BCPI10 with linolenic acid domination (∼66%) followed by linoleic acid (∼19%) and oleic acid (∼6%), contributing PUFAs (∼86%). Optimized protein isolates, black (BCPI10) and white (WCPI10) chia, had shown purity, L*-value, solubility, and yields of 90.65%, 75.86%, 77.75%, 11.30%, and 90.00%, 77.83%, 76.07%, 10.69%, respectively. BCPI10 depicted higher EAA (33.19 g/100 g N) and EEA indices (57.676%) compared to WCPI10 (32.14 g/100 g N) and 56.360%, respectively. Amino acid profiling indicated higher, PER, TAA, TEAA, TNEAA, TAAA, TBA, acidic AA values for BCPI10, and higher leucine/isoleucine ratio for WCPI10 having leucine and sulfur amino acids as limiting amino acids. BCPI10 had higher sulfur-containing amino acid contents, as the main contributor to the albumin a water-soluble fraction, leading to its higher in vitro digestibility (71.97%) than WCPI10 (67.70%). Both isolates exhibited good WHC and OHC of 3.18, 2.39 and 3.00, 2.20, respectively. Both protein isolates had similar ∆Td (°C) values with some variation in FTIR spectrum from 1000 cm-1 to 1651 cm-1 having more peak intensity for BCPI10. SDS-PAGE indicated bands at 150 kDa, representing globulin and mild bands at 25-33 kDa for glutelin and albumin. A significant (p < 0.05) variation reported in this study for protein and lipid profiles of both genotype attributes to genetic differences between the seeds. PRACTICAL APPLICATION: Based on the nutritional profile, both chia seed isolates (black and white) are suitable for consumption with an edge for black seed when supplemented with their limiting amino acids. The high values of the functional properties and structural characteristics combined with high nutritional values make the chia protein isolate an excellent source of raw material for various food formulations. Fatty acid profile of the oils from the genotypes showed the presence of high amounts of unsaturated fatty acids, especially the PUFAs with more number of fatty acids in black chia seed. The excellent lipid profile of chia seed oil indicates the benefit of using chia seed oil as a source of essential fatty acids in the human diet for optimal health.

Bioavailability of Tauropine After Oral Ingestion in Mouse.

In the present study, we investigated the pharmacokinetics of oral ingested tauropine which is a natural taurine derivative found in marine invertebrates, such as abalone, and in mouse. To measure tauropine in the blood, it was derivatized with phenyl isothiocyanate (PITC), and PITC-tauropine was separated by reverse-phase high-performance liquid chromatography (HPLC) and detected by ultraviolet absorbance. Tauropine was detectable in the blood obtained from mice intraperitoneally injected with tauropine. However, it was not detectable in blood obtained from orally treated mice. In conclusion, oral ingested tauropine may be poorly absorbed by the gastrointestinal tract and transported into the blood.

Development of soybean experimental lines with enhanced protein and sulfur amino acid content.

Soybean is the preferred protein source for both poultry and swine feed. However, this preferred status is being challenged due to competition from alternative feed ingredients. To overcome this, it becomes necessary for breeders to develop soybean cultivars that contain higher protein and better nutritional composition. In this study, we have developed experimental soybean lines that not only contain significantly higher amounts of protein but also improved sulfur amino acid content. This objective was achieved by crossing a O-acetylserine sulfhydrylase (OASS) overexpressing transgenic soybean line with elevated levels of sulfur amino acid content (CS) with a high protein Korean soybean cultivar (Lee 5). Introgression of high protein and overexpression of OASS was monitored in the experimental lines at each successive generation (F2-F6) by measuring protein content and OASS activity. The average protein content of transgenic CS and Lee 5 seeds were 34.8 % and 44.7 %, while in the experimental soybean lines the protein content ranged from 41.3 %-47.7 %, respectively. HPLC and inductively coupled plasma-mass spectrometry analyses revealed that all the experimental lines developed in this study contained significantly higher amounts of sulfur containing amino acids and elemental sulfur in the seeds. The sulfur amino acid (cysteine + methionine) content of the experimental lines ranged from 1.1 % to 1.26 % while the parents Lee 5 and CS had 0.79 % and 1.1 %, respectively. SDS-PAGE and western blot analysis demonstrated that the accumulation of Bowman-Birk protease inhibitor and lunasin, two sulfur amino acid rich peptides, were elevated in experimental soybean lines. High-resolution 2D-gel electrophoresis and Delta2D gel analysis validated that an overall increase in the different subunits of 7S ß-conglycinin and 11S glycinin were mainly responsible for the observed increase in the total amount of protein in experimental lines.

An efficient method for the determination of enantiomeric purity of racemic amino acids using micellar chromatography, a green approach.

A simple, sensitive and green micellar liquid chromatographic method (RP-HPLC) was developed for enantioseparation of four racemic amino acids, namely, (RS)-selenomethionine, (RS)-methionine, (RS)-cysteine and (RS)-penicillamine. An aqueous solution of sodium dodecyl sulfate and Brij-35 was prepared and used as mobile phase for HPLC analysis. Activated esters of (S)-ibuprofen, (S)-ketoprofen and (S)-levofloxacin were synthesized by reacting them with N-hydroxybenzotriazole. These esters were characterized by UV, IR, 1 HNMR, HRMS and elemental analysis. These chiral reagents (activated esters) were used for the synthesis of diastereomeric derivatives of the chosen amino acids. The diastereomeric derivatives were separated on a C18 column by micellar liquid chromatography. Chromatographic conditions were optimized by varying concentration of surfactant in aqueous solution, and by varying the concentration and pH of the buffer. The green assessment score was calculated for the developed method (78, an excellent green method score). In addition, density functional theory calculations were performed, using Gaussian 09 rev. A.02 and hybrid density functional B3LYP with a 6-31G* basis set program, in order to develop lowest energy optimized structures of diastereomeric derivatives. The method was validated according to International Conference on Harmonization guidelines and the retention factor (k), selectivity factor (α), resolution factor (RS ) and limit of detection (0.295 ng ml-1 ) and limit of quantification (0.896 ng ml-1 ) were calculated.

Metabolomic Analysis of Liver from Dietary Biotin Deficient Mice.

Biotin is a water-soluble B-complex vitamin that functions as a cofactor of five carboxylases. Because biotin-dependent carboxylases catalyze indispensable cellular metabolic functions, biotin deficiency is considered to be involved in various pathological conditions. Moreover, biotin supplementation shows pharmacological effects in vivo. However, the precise mechanisms by which biotin deficiency induces pathological conditions remain unclear. Although abnormal metabolites are used as indicators for biotin deficiency, few comprehensive analyses of total metabolites have been reported. In this study, we analyzed the metabolomic profiles of liver extracts prepared from biotin-sufficient (BS) and -deficient (BD) mice. Thirteen of 126 metabolites showed significantly different concentrations between liver extracts from BD and BS mice. The concentrations of 5 essential amino acids, Met, Val, Thr, Ile, and Leu, and 2 conditionally essential amino acids, Cys and Tyr were significantly lower in BD mice than in BS mice. Among these, the concentrations of sulfur-containing amino acids, Cys and Met, were more than 1.5-fold lower in BD mice. The concentrations of Met metabolites, such as S-adenosylmethionine and S-adenosylhomocysteine were not significantly different between the two groups. The concentrations of glutathione and its reaction intermediates γ-Glu-Cys tendency to be lower in BD mice. The present study revealed that biotin deficiency induces an abnormal amino acids composition, especially among sulfur-containing amino acids and provide important information on the effect of biotin as a pharmacological agent.

Distribution and possible biosynthetic pathway of non-protein sulfur amino acids in legumes.

Some grain legumes store sulfur in the form of non-protein amino acids in seed. γ-Glutamyl-S-methylcysteine is found in Phaseolus and several Vigna species. γ-Glutamyl-S-ethenylcysteine, an antinutritional compound, is present in Vicia narbonensis. In P. vulgaris, free S-methylcysteine levels are higher at early stages of seed development followed by a decline. γ-Glutamyl-S-methylcysteine accumulates later, in two phases, with a lag during reserve accumulation. The concentration of total S-methylcysteine, quantified after acid hydrolysis, is positively regulated by sulfate nutrition. The levels of both γ-glutamyl-S-methylcysteine and γ-glutamyl-S-ethenylcysteine are modulated in response to changes in seed protein composition. A model is proposed whereby the majority of γ-glutamyl-S-methylcysteine in P. vulgaris is synthesized via the intermediate S-methylhomoglutathione. Knowledge of the biosynthesis of non-protein sulfur amino acids is required for metabolic engineering approaches, in conjunction with manipulation of the protein sink, to increase the concentration of nutritionally essential methionine and cysteine. This would improve protein quality of some important legume crops.

Effect of incremental levels of dietary total sulfur amino acids on performance of white leghorn laying hens fed diets containing guar (Cyamopsis tetragonoloba) meal.

BACKGROUND: The present study evaluated the potential of increasing dietary levels of the most limiting amino acids such as total sulfur amino acids (TSAA) with respect to mitigating the adverse effects of the inclusion of guar meal (GM) in the diet of WL layers on egg production, feed efficiency and egg weight. In total, 4928 WL (Lohmann L) hens (32 week of age) were randomly allotted to eight treatments with seven replicates of 88 birds. The dietary treatments were a set of four maize-soybean meal based diets containing four graded concentrations of TSAA (5.85, 6.18, 6.51 and 6.84 g kg-1 ) and another set of four diets containing 100 g kg-1 guar meal (GM) with similar concentrations of TSAA. RESULTS: The inclusion of 100 g kg-1 GM in the diet caused a 20%, 23%, 35.5% and 6.7% reduction in egg production, daily egg mass, feed efficiency and egg weight, respectively, when the diet contained 5.85 g kg-1 TSAA. Increasing TSAA levels in GM groups reduced the adverse effects on egg production and feed efficiency 50%, with no effect on egg weight. The TSAA requirement of WL birds fed 10% GM was estimated as 6.28-7.39 g kg-1 diet at different ages. CONCLUSION: Increasing dietary TSAA to 7.39, 6.28, 6.53 and 6.37 g kg-1 or dTSAA (digestible TSAA) to 6.53, 5.32, 5.80 and 5.63 g kg-1 during 33-36, 37-40, 41-44 and 45-48 weeks of age, respectively, may be an effective and practical strategy for reducing the adverse effects of inclusion of 100 g kg-1 GM in the diet of WL layers. © 2018 Society of Chemical Industry.

Quantification of sulphur amino acids by ultra-high performance liquid chromatography in aquatic invertebrates.

We examined the performance of an ultra-high performance liquid chromatography method to quantify protein-bound sulphur amino acids in zooplankton. Both cysteic acid and methionine sulfone were linear from 5 to 250 pmol (r2 = 0.99), with a method detection limit of 13 pmol and 9 pmol, respectively. Although there was no matrix effect on linearity, adjacent peaks and co-eluting noise from the invertebrate proteins increased the detection limits when compared to common standards. Overall, performance characteristics were reproducible and accurate, and provide a means for quantifying sulphur amino acids in aquatic invertebrates, an understudied group.

Colorimetric detection of biothiols based on aggregation of chitosan-stabilized silver nanoparticles.

We have described a simple and reliable colorimetric method for the sensing of biothiols such as cysteine, homocysteine, and glutathione in biological samples. The selective binding of chitosan capped silver nanoparticles to biothiols induced aggregation of the chitosan-Ag NPs. But the other amino acids that do not have thiol group cannot aggregate the chitosan-Ag NPs. Aggregation of chitosan-Ag NPs has been confirmed with UV-vis absorption spectra, zeta potential and transmission electron microscopy images. Under optimum conditions, good linear relationships existed between the absorption ratios (at A500/A415) and the concentrations of cysteine, homocysteine, and glutathione in the range of 0.1-10.0µM with detection limits of 15.0, 84.6 and 40.0nM, respectively. This probe was successfully applied to detect these biothiols in biological samples (urine and plasma).

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near-infrared laser-induced fluorescence detection using a new BODIPY-based probe as labeling reagent.

A CZE with near-infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ-glutamylcysteine, cysteinylglycine, and N-acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N-acetylcysteine to 0.31 nmol/L for γ-glutamylcysteine, which are better than or comparable to those reported with other derivatization-based CE-LIF methods. As the first trial of NIR CE-LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5-104.3%.

Alterations in sulfur amino acid metabolism in mice treated with silymarin: a novel mechanism of its action involved in enhancement of the antioxidant defense in liver.

It has been known that silymarin exhibits protective activity against oxidative liver injury induced by various hepatotoxicants, but the underlying mechanism of its beneficial action remains unclear. We determined the alterations in sulfur-containing amino acid metabolism induced by silymarin in association with its effects on the antioxidant capacity of liver. Male mice were treated with silymarin (100 or 200 mg/kg, p. o.) every 12 h for a total of 3 doses, and sacrificed 6 h after the final dosing. The hepatic methionine level was increased, but the activity and protein expression of methionine adenosyltransferase were decreased by silymarin in a dose-dependent manner. S-Adenosylmethionine or homocysteine concentration was not changed, whereas the sulfur-containing metabolites generated from homocysteine in the transsulfuration pathway including cystathionine, cysteine, and glutathione were increased significantly. Cystathionine ß-synthase was induced, but cysteine dioxygenase was downregulated, both of which would contribute to the elevation of cysteine and its product, glutathione, in liver. Oxygen radical scavenging capacity of liver cytosol against peroxyl radical and peroxynitrite was increased, and also hepatic lipid peroxidation was diminished in the silymarin-treated mice. Taken together, the results demonstrate that silymarin enhances hepatic glutathione generation by elevating cysteine availability via an increment in cysteine synthesis and an inhibition of its catabolism to taurine, which may subsequently contribute to the antioxidant defense of liver.

Determination of aminothiols by liquid chromatography with amperometric detection at a silver electrode: application to white wines.

Liquid chromatography coupled to a silver electrode based flow-through amperometric detector (LC-EC-Ag) was developed for the determination of aminothiols in white wines. The C18 reversed phase LC system operated in the isocratic mode at 0.7 mL min(-1) and used an acidic mobile phase composed of formic acid, EDTA, sodium nitrate, sodium hydroxide, and methanol 1% (v/v) at pH 4.5. The working electrode operated at 0.08 V vs Ag/AgCl, 3M KCl and its manual cleaning was realized once a month by smoothing on a polishing cloth. The analyzed aminothiols were resolved and eluted within 4 min, and all standard curves were linear in the range 2×10(-7)-2×10(-5) M. The analyzed wine samples needed no preparation other than dilution with the mobile phase. The concentration of cysteine (CYS), homocysteine (HCYS), glutathione (GSH) and N-acetylcysteine (NAC) in bottled white wines, determined by the method of standard addition, was found to be in the low µM range (0.2-2 mg L(-1)) depending on the wine type and its age.

Monitoring the production process of selenized yeast by elemental speciation analysis.

Elemental speciation analysis was implemented as an essential tool set addressing optimum fermentation conditions for the production of selenized yeast feed supplements. Accordingly, the study addressed intracellular levels of (1) total selenium and sulfur, (2) seleno methionine (SeMet), (3) cysteine (Cys) and methionine (Met) and (4) selenite and selenate. Dedicated sample preparation- and LC-ICP-MS methods were implemented and validated using the reference material Selm-1. Excellent repeatability precisions <10% (n = 4 biological replicates) could be obtained for all parameters. The study comprised fermentation monitoring over 72 hours (6 different time points) for a Saccharomyces cerevisiae strain under different selenite feed conditions. It was observed that for this strain an increase in the selenium concentration in the fermentation feed by 50% did not result in enhanced selenium accumulation. Fermentation monitoring of three different Saccharomyces cerevisiae strains under the same conditions showed strain specific selenium uptake after 72 hours. The strain with the lowest cell viability of 60% showed the lowest SeMet content. After 47 h of fermentation, all strains reached a critical point, at which seleno methionine accounted for approximately 100% of the total selenium and cell viability started to decrease. This could be explained by sulfur limitation and/or excess of the seleno methionine storage capacity. Strains showing cell viability of approx. 90% after 72 hours of fermentation revealed SeMet concentrations up to 3000 µg g(-1). In the final product, an apparent threshold level for Met/SeMet of approx. 1 was observed for all strains.

Fluorosurfactant-prepared triangular gold nanoparticles as postcolumn chemiluminescence reagents for high-performance liquid chromatography assay of low molecular weight aminothiols in biological fluids.

Our recent study demonstrates the synthesized triangular gold nanoparticles (AuNPs) by trisodium citrate reduction of HAuCl(4) in the presence of nonionic fluorosurfactant (FSN) could display stronger catalytic activity towards luminol-chemiluminescence (CL) than spherical AuNPs. Ultratrace aminothiols may cause a great decrease in CL intensity of the triangular AuNPs-luminol CL system. In this article, we utilize the as-prepared triangular AuNPs as novel postcolumn CL reagents to explore a simple high-performance liquid chromatography (HPLC)-CL method for the determination of low molecular weight aminothiols (i.e., cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine). The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and highly selective and sensitive towards reduced aminothiols. The detection limits at a signal-to-noise ratio of 3 for cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine were 0.016, 0.08, 0.1, 0.04 and 0.1pmol, respectively. Recoveries from spiked urine and plasma samples were 95.7-104.3%. The applicability of the proposed method has been validated by determining these low molecular weight aminothiols in human urine and plasma samples with satisfactory results, and thus it will have great potential application in clinical diagnosis.

Simultaneous determination of ascorbic acid, aminothiols, and methionine in biological matrices using ion-pairing RP-HPLC coupled with electrochemical detector.

A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution((aqueous)). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution((aqueous)) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C(18) (250 × 4.6 mm, 5 µm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.

The oxidized thiol proteome in fission yeast--optimization of an ICAT-based method to identify H2O2-oxidized proteins.

Major intracellular disulfide formation is prevented in the cytosol by potent reducing systems. However, protein thiols can be oxidized as a consequence of redox-mediated physiological reactions or due to the unwanted toxicity of reactive oxygen species. In addition, the reactivity of cysteine residues towards peroxides is used by H(2)O(2) sensors in signal transduction pathways in a gain-of-function process to induce transcriptional antioxidant responses. Thus, the Schizosaccharomyces pombe peroxiredoxin Tpx1 and the transcription factor Pap1 are sensors of H(2)O(2) meant to promote cell survival. In an attempt to compare signaling events versus global thiol oxidation, we have optimized thiol-labeling approaches to characterize the disulfide proteome of fission yeast in response to added H(2)O(2). We propose a method based on (i) freezing the redox state of thiols with strong acids prior to cell lysis; (ii) blocking thiol groups with iodoacetamide, and reversibly oxidized thiols with heavy and light isotope-coded affinity tags (ICAT) reagents; and (iii) quantifying individual relative protein concentrations with stable-isotope dimethyl labeling. We have applied this highly sensitive strategy to provide a map of H(2)O(2)-dependent oxidized thiols in fission yeast, and found Tpx1 and Pap1 as some of the major targets.

[Determination of sulfur amino acids in feedstuffs by high performance liquid chromatography coupled with pre-column derivatization].

A method of quantitative analysis of sulfur amino acids in feedstuffs by high performance liquid chromatography coupled with pre-column derivatization was developed. Before the feedstuffs were hydrolyzed under acidic condition, the cystine and methionine in the feedstuffs were oxidized to cysteic acid and methionine sulfone respectively by performic acid, and then derivatized by 2,4-dinitrofluorobenzene. The separation was performed on an Elite AAK C18 column (250 mm x 4.6 mm, 5 microm) by the gradient elution of 0.05 mol/L sodium acetate and acetonitrile-water (50: 50, v/v) as the mobile phase with a flow rate of 1.2 mL/min at 31 degrees C. The detection wavelength was set at 360 nm. The linearities of cystine and methionine were good in the ranges of 0.4 - 16.0 mg/L and 0.7 - 29.6 mg/L with the correlation coefficients of 0.999 9 and 0.999 8, respectively. The quantification limits (S/N = 10) were 2.6 microg/kg, 3.1 microg/kg, and the recoveries were 100.28% - 102.00% and 105.72% - 107.89%, respectively. The method can be adapted to the accurate quantification of the sulfur amino acids in feedstuffs.

Thiol redox proteomics identifies differential targets of cytosolic and mitochondrial glutaredoxin-2 isoforms in Saccharomyces cerevisiae. Reversible S-glutathionylation of DHBP synthase (RIB3).

Yeast Grx2 plays a role in the antioxidant glutathione linked defense acting on the redox status of protein cysteines, but the exact action or its specificity is not known. Moreover, it localizes in cytosol and mitochondria where it can exert different functions. To search for functions of Grx2 we determined the differential "Thiolic Redox Proteome" of control and peroxide-treated yeast mutant cells lacking the gene for Grx2 or expressing Grx2 exclusively in the mitochondria. Forty-two proteins have been identified that have alternative redox oxidation states as a consequence of Grx2 absence from the cell or expression in the mitochondria and absence from the cytosol. The precise cysteine residues affected have been mapped for each protein. One target protein, Rib3p, which has as yet an undefined function in respiration, was confirmed to have its Cys56 reversibly S-glutathionylated in vitro in a Grx2p dependent process. Grx2-dependent redox changes in key enzymes of glutamate consuming amino acid biosynthetic pathways could favor glutathione biosynthesis. Other target proteins are involved in membrane fusion, cell wall structure and ribosome assembly, but others are of unknown function. These results provide clues on the metabolic hot spots of redox regulatory mechanisms.