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Uwe Karst - One of the best experts on this subject based on the ideXlab platform.
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simultaneous lc ms ms determination of Thiols and disulfides in urine samples based on differential labeling with ferrocene based maleimides
Analytical Chemistry, 2007Co-Authors: Bettina Seiwert, Uwe KarstAbstract:A method for the simultaneous determination of a series of Thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound Thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free Thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound Thiols by tris(2-carboxyethyl)phosphine, the newly formed Thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further Thiols. The method was successfully applied to the analysis of free and disulfide-bound Thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of Thiols in urine samples. The Thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.
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simultaneous lc ms ms determination of Thiols and disulfides in urine samples based on differential labeling with ferrocene based maleimides
Analytical Chemistry, 2007Co-Authors: Bettina Seiwert, Uwe KarstAbstract:A method for the simultaneous determination of a series of Thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound Thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free Thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound Thiols by tris(2-carboxyethyl)phosphine, the newly formed Thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further Thiols. The method was successfully applied to the analysis of free and disulfide-bound Thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range...
Bettina Seiwert - One of the best experts on this subject based on the ideXlab platform.
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simultaneous lc ms ms determination of Thiols and disulfides in urine samples based on differential labeling with ferrocene based maleimides
Analytical Chemistry, 2007Co-Authors: Bettina Seiwert, Uwe KarstAbstract:A method for the simultaneous determination of a series of Thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound Thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free Thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound Thiols by tris(2-carboxyethyl)phosphine, the newly formed Thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further Thiols. The method was successfully applied to the analysis of free and disulfide-bound Thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of Thiols in urine samples. The Thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.
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simultaneous lc ms ms determination of Thiols and disulfides in urine samples based on differential labeling with ferrocene based maleimides
Analytical Chemistry, 2007Co-Authors: Bettina Seiwert, Uwe KarstAbstract:A method for the simultaneous determination of a series of Thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound Thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free Thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound Thiols by tris(2-carboxyethyl)phosphine, the newly formed Thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further Thiols. The method was successfully applied to the analysis of free and disulfide-bound Thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range...
Lingliang Long - One of the best experts on this subject based on the ideXlab platform.
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a sensitive and selective fluorescent Thiol probe in water based on the conjugate 1 4 addition of Thiols to α β unsaturated ketones
Chemistry: A European Journal, 2009Co-Authors: Lin Yuan, Yanming Feng, Lingliang LongAbstract:Shedding light on Thiol detection: A compound (see scheme) was developed as a novel, highly sensitive and selective fluorescent Thiol probe, which also features suitable water solubility, functions rapidly under neutral conditions, and has excitation and emission in the visible region. Thus, it may be useful for potential biological applications. Compound 1 was designed and synthesized as a new fluorescent Thiol probe. Probe 1 was constructed on the basis of the conjugate 1,4-addition of Thiols to α,β-unsaturated ketones. Notably, probe 1 has suitable water solubility, which allows the sensing assay to be performed in water. Probe 1 is highly sensitive for Thiols with a 211-fold fluorescence dynamic range and a low detection limit of 9.25×10−7 M. The major features of probe 1 also include a high selectivity for Thiols over other relevant biological species, excitation and emission in the visible region, rapid functioning at pH 7.4, and a good linear relationship between the fluorescence signal and the Thiol concentration. Accordingly, these desirable characteristics may render probe 1 as potentially useful for biological applications.
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a sensitive and selective fluorescent Thiol probe in water based on the conjugate 1 4 addition of Thiols to α β unsaturated ketones
Chemistry: A European Journal, 2009Co-Authors: Weiying Lin, Lin Yuan, Yanming Feng, Zengmei Cao, Lingliang LongAbstract:Compound 1 was designed and synthesized as a new fluorescent Thiol probe. Probe 1 was constructed on the basis of the conjugate 1,4-addition of Thiols to alpha,beta-unsaturated ketones. Notably, probe 1 has suitable water solubility, which allows the sensing assay to be performed in water. Probe 1 is highly sensitive for Thiols with a 211-fold fluorescence dynamic range and a low detection limit of 9.25x10(-7) M. The major features of probe 1 also include a high selectivity for Thiols over other relevant biological species, excitation and emission in the visible region, rapid functioning at pH 7.4, and a good linear relationship between the fluorescence signal and the Thiol concentration. Accordingly, these desirable characteristics may render probe 1 as potentially useful for biological applications.
Chris J Hamilton - One of the best experts on this subject based on the ideXlab platform.
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Physiological Studies of Chlorobiaceae Suggest that BacilliThiol Derivatives Are the Most Widespread Thiols in Bacteria
American Society for Microbiology, 2018Co-Authors: Jennifer Hiras, Chris J Hamilton, Sunil V. Sharma, Vidhyavathi Raman, Ryan A. J. Tinson, Miriam Arbach, Dominic F. Rodrigues, Javiera Norambuena, Thomas E. HansonAbstract:Low-molecular-weight Thiols are key metabolites that participate in many basic cellular processes: central metabolism, detoxification, and oxidative stress resistance. Here we describe a new Thiol, N-methyl-bacilliThiol, found in an anaerobic phototrophic bacterium and identify a gene that is responsible for its synthesis from bacilliThiol, the main Thiol metabolite in many Gram-positive bacteria. We show that the presence or absence of this gene in a sequenced genome accurately predicts Thiol content in distantly related bacteria. On the basis of these results, we analyzed genome data and predict that bacilliThiol and its derivatives are the most widely distributed Thiol metabolites in biology.Low-molecular-weight (LMW) Thiols mediate redox homeostasis and the detoxification of chemical stressors. Despite their essential functions, the distribution of LMW Thiols across cellular life has not yet been defined. LMW Thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae. Here we show that Chlorobaculum tepidum synthesizes a novel LMW Thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C14H24N2O10S, which suggests that the new LMW Thiol is closely related to bacilliThiol (BSH), the major LMW Thiol of low-G+C Gram-positive bacteria. The Cba. tepidum LMW Thiol structure was N-methyl-bacilliThiol (N-Me-BSH), methylated on the cysteine nitrogen, the fourth instance of this modification in metabolism. Orthologs of bacilliThiol biosynthetic genes in the Cba. tepidum genome and the CT1040 gene product, N-Me-BSH synthase, were required for N-Me-BSH synthesis. N-Me-BSH was found in all Chlorobiaceae examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes. A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobiaceae, Bacteroidetes, Deinococcus-Thermus, and Firmicutes but also by Acidobacteria, Chlamydiae, Gemmatimonadetes, and Proteobacteria. Thus, BSH and derivatives appear to be the most broadly distributed LMW Thiols in biology
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Biophysical features of bacilliThiol, the glutathione surrogate of Bacillus subtilis and other firmicutes.
Chembiochem : a European journal of chemical biology, 2013Co-Authors: Sunil Sharma, Miriam Arbach, Alexandra A. Roberts, Colin J. Macdonald, Murree Groom, Chris J HamiltonAbstract:BacilliThiol (BSH) is the major low-molecular-weight (LMW) Thiol in many low-G+C Gram-positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW Thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycoThiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW Thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW Thiols in terms of its Thiol pKa, redox potential and Thiol–disulfide exchange reactivity. Both the Thiol pKa and the standard Thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in Thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM, which is significantly greater than previously observed from single measurements taken during mid-exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.
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low molecular weight Thiols in Thiol disulfide exchange
Antioxidants & Redox Signaling, 2013Co-Authors: Koen Van Laer, Chris J HamiltonAbstract:Abstract Significance: Oxidative stress is widely invoked in inflammation, aging, and complex diseases. To avoid unwanted oxidations, the redox environment of cellular compartments needs to be tightly controlled. The complementary action of oxidoreductases and of high concentrations of low-molecular-weight (LMW) nonprotein Thiols plays an essential role in maintaining the redox potential of the cell in balance. Recent Advances: While LMW Thiols are central players in an extensive range of redox regulation/metabolism processes, not all organisms use the same Thiol cofactors to this effect, as evidenced by the recent discovery of mycoThiol (MSH) and bacilliThiol (BSH) among different gram-positive bacteria. Critical Issues: LMW Thiol–disulfide exchange processes and their cellular implications are often oversimplified, as only the biology of the free Thiols and their symmetrical disulfides is considered. In bacteria under oxidative stress, especially where concentrations of different LMW Thiols are comparab...
Xiangming Guan - One of the best experts on this subject based on the ideXlab platform.
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determination of Thiols and disulfides via hplc quantification of 5 thio 2 nitrobenzoic acid
Journal of Pharmaceutical and Biomedical Analysis, 2008Co-Authors: Wei Chen, Yong Zhao, Teresa Seefeldt, Xiangming GuanAbstract:This work presents an assay for total Thiols and total disulfides in biological samples via HPLC quantification of 5-thio-2-nitrobenzoic acid (TNB) derived from the reaction of Thiols with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent). This method also provides simultaneous quantification of glutathione (GSH) via the measurement of the GSH-DTNB adduct (GSH-TNB). By using 326nm as the detecting wavelength, the HPLC detection limit for TNB and the GSH-TNB adduct was determined to be 15 and 7.5pmol respectively. A recovery study with OVCAR-3 cells revealed that the recovery yields for TNB in the procedures for determining non-protein Thiols, protein Thiols, non-protein disulfides, and protein disulfides were 99.4+/-1.2% (n=3), 98.1+/-5.0% (n=3), 95.6+/-0.9% (n=3), and 96.6+/-2.3% (n=3) respectively. The recovery yield for GSH-TNB in the procedures for determining non-protein Thiols, protein Thiols, non-protein disulfides, and protein disulfides was 99.0+/-0.3% (n=3), 95.1+/-4.9% (n=3), 96.8+/-0.6% (n=3), and 95.1+/-2.9% (n=3) respectively. The reproducibility, expressed as the relative standard deviation for the analyte, for TNB was determined to be 2.8% (n=6) for non-protein Thiols, 3.9% (n=6) for protein Thiols, 3.6% (n=6) for non-protein disulfides and 4.6% (n=6) for protein disulfides. The reproducibility for GSH-TNB was determined to be 1.6% (n=6) for non-protein Thiols and 2.6% (n=6) for non-protein disulfides. By comparing the amount of GSH determined in a biological sample before NaBH(4) reduction with that after the reduction, this method can provide information associated with Thiol glutathionylation which would be useful for protein glutathionylation study. This method should be applicable to cellular, subcellular, protein, or other biomatrix samples for Thiol and disulfide quantification and will be a useful analytical method in the study of Thiol redox state and Thiol glutathionylation.
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a simultaneous liquid chromatography mass spectrometric assay of glutathione cysteine homocysteine and their disulfides in biological samples
Journal of Pharmaceutical and Biomedical Analysis, 2003Co-Authors: Xiangming Guan, Brianna N Hoffman, Chandradhar Dwivedi, Duane P MattheesAbstract:A liquid chromatography/mass spectrometric (LC/MS) method was developed for simultaneous detection and quantitation of glutathione (GSH), glutathione disulfide (GSSG), cysteine (CysSH), homocysteine (HCysSH) and homocystine in biological samples (rat brain, lung, liver, heart, kidneys, erythrocytes and plasma). Thiols were derivatized with a large excess of Ellman's reagent, a Thiol-specific reagent, to ensure an instantaneous and complete derivatization. The derivatization blocked the oxidation of the Thiols to disulfides, preventing errors caused by Thiol oxidation. The samples were then analyzed by LC/MS. The method provides a highly selective and sensitive assay for these endogenous Thiols and their corresponding disulfides. The detection limits for GSH, GSSG, CysSH, HCysSH and homocystine were 3.3, 3.3, 16.5, 29.6 and 14.9 pmol, respectively. An attempt for cystine analysis was unsuccessful due to earlier elution of the compound and strong interferences caused by other endogenous compounds. This method will be a useful tool in the investigation of the roles of these important Thiol-containing compounds and their corresponding disulfides in physiological and pathological processes.
