The Experts below are selected from a list of 183 Experts worldwide ranked by ideXlab platform
Shuhui Chen - One of the best experts on this subject based on the ideXlab platform.
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identification of cytosolic protein targets of Catechol Estrogens in breast cancer cells using a click chemistry based workflow
Journal of Proteome Research, 2021Co-Authors: Tingen Huang, Yichen Liu, Junghsiang Tai, Shuhui ChenAbstract:Catechol Estrogens (CEs) are known to be toxic metabolites and the initiators of the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins, but their cellular protein targets remain unexplored. Here, we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an improved click chemistry-based workflow. Multiple comparative proteomics composed of an experimental pair (probe versus solvent) and two control pairs (solvent versus solvent and probe versus solvent without enrichment) were studied using stable isotope dimethyl labeling. The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free linker coupled with on-bead digestion and redigestion of the proteins cleaved from the beads was shown to greatly improve the recovery and identification of CE-adducted peptides. A total of 310 protein targets and 40 adduction sites were repeatedly (n ≥ 2) identified with D/H (probe/solvent) ratio >4 versus only one identified with D/H >4 from the two control pairs, suggesting that our workflow imposes only a very low background. Meanwhile, multiple comparative D/H ratios revealed that CEs may downregulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CE-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data will provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.
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targeting endogenous adduction level of serum albumin by parallel reaction monitoring via standard additions and intact protein measurement biological dosimetry of Catechol Estrogens
Analytical Chemistry, 2019Co-Authors: Yu Shan Huang, Hsin Chen, Tingen Huang, Yu Min Lin, Chong Hua Syu, Shuhui ChenAbstract:Abundant blood proteins adducted by active electrophiles are excellent markers to predict the risk of electrophile-induced toxicity. However, detecting endogenously adducted proteins by bottom-up selective (or parallel) reaction monitoring (SRM/PRM) is challenging because of the high variability in sample preparation and detection as well as low adduction levels. Here, we reported a new approach in developing PRM methods by combining intact protein measurement with standard additions to target optimal conditions for detecting Catechol Estrogens (CEs)-adducted human serum albumin (HSA). Blood serum was added with multiple amounts of CEs to obtain serum standards. Intact protein measurement revealed two linear ranges of adduction levels (adducted-CE/HSA): 0.34-0.42 (R2 > 0.94) and 0.81-8.54 (R2 > 0.96) against the amount of added CEs, respectively. Six adduction sites were identified by trypsin (K20, C34, K73, K281, H338, K378) or chymotrypsin (K20, C34, K378) digestion. PRM methods targeting all adducted/nonadducted peptide pairs based on chymotrypsin or trypsin digestion were developed, and the data were compared with those obtained by intact protein measurement. Correlation plots indicated that chymotrypsin-PRM leads to poor sensitivity and largely underestimated protein adduction levels. Trypsin-PRM leads to sensitive and highly correlated (R2 > 0.91) protein adduction levels with a detection limit below the endogenous level and relative standard deviation <25%. As a proof of concept, clinical serum samples were examined by trypsin-PRM, and a slightly higher adduction level was observed for the obesity group when compared with the healthy group. This is the first report on determining adduction levels of blood proteins for long-term exposure to CEs. The standard addition approach can be generally applied to protein adductomics with resolvable mass increments by intact protein measurement to accelerate the development of bottom-up methods close to the inherent limit.
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in situ click reaction coupled with quantitative proteomics for identifying protein targets of Catechol Estrogens
Journal of Proteome Research, 2018Co-Authors: Hueichen Liang, Yichen Liu, Hsin Chen, Chihyen Wang, Shun Fen Tzeng, Shuhui ChenAbstract:Catechol Estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography–mass spectrometry (LC–MS). A total of 334 liver proteins were repeatedly identified (n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific b...
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In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens
2018Co-Authors: Hueichen Liang, Yichen Liu, Hsin Chen, Chihyen Wang, Shun Fen Tzeng, Shuhui ChenAbstract:Catechol Estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography–mass spectrometry (LC–MS). A total of 334 liver proteins were repeatedly identified (n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, Catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems
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site specific covalent modifications of human insulin by Catechol Estrogens reactivity and induced structural and functional changes
Scientific Reports, 2016Co-Authors: Chieh Ming Fang, Hueichen Liang, Juei Tang Cheng, Tzufan Wang, Chiao Chen Chen, Junghsiang Tai, Shuhui ChenAbstract:Proteins, covalently modified by Catechol Estrogens (CEs), were identified recently from the blood serum of diabetic patients and referred to as estrogenized proteins. Estrogenization of circulating insulin may occur and affect its molecular functioning. Here, the chemical reactivity of CEs towards specific amino acid residues of proteins and the structural and functional changes induced by the estrogenization of insulin were studied using cyclic voltammetry, liquid chromatography-mass spectrometry, circular dichroism spectroscopy, molecular modeling, and bioassays. Our results indicate that CEs, namely, 2- and 4-hydroxyl Estrogens, were thermodynamically and kinetically more reactive than the Catechol moiety. Upon co-incubation, intact insulin formed a substantial number of adducts with one or multiple CEs via covalent conjugation at its Cys 7 in the A or B chain, as well as at His10 or Lys29 in the B chain. Such conjugation was coupled with the cleavage of inter-chain disulfide linkages. Estrogenization on these sites may block the receptor-binding pockets of insulin. Insulin signaling and glucose uptake levels were lower in MCF-7 cells treated with modified insulin than in cells treated with native insulin. Taken together, our findings demonstrate that insulin molecules are susceptible to active estrogenization, and that such modification may alter the action of insulin.
Yoshifumi Matsuda - One of the best experts on this subject based on the ideXlab platform.
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hplc for stress free screening of potential prostate cancer marker Catechol Estrogens in urine using a diamond electrode electrochemical and a fluorescence detector
Journal of Separation Science, 2007Co-Authors: Masatoki Katayama, Kiyoshi Takamatsu, Satoru Kaneko, Keisuke Miyaji, Hiromichi Ishikawa, Yoshifumi MatsudaAbstract:Improvement of the sensitivity and specificity of a simultaneous stress-free screening method for Catechol Estrogens as a potential prostate cancer marker in urine has been accomplished by HPLC with a diamond-electrode electrochemical detector and a fluorescence detector. Since taking urine samples generates less stress (or pain) than the drawing of blood, the method can readily be applied to almost any patient, and will also assist in improving the sensitivity and specificity of the prostatic specific antigen test. Catechol Estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestriol) and Estrogens (estrone, estradiol, estriol) were separated on an Inertsil ODS-II column with acetonitrile-potassium dihydrogen phosphate (pH 3.0). The diamond-electrode electrochemical detector used had the great advantage of being a maintenance-free system, and could sequentially analyze hundreds of samples. Fluorescence detection improved the sensitivity 10-500 times (e. g., the LOD of 2-hydroxyestriol was improved 250 times) compared to previous electrochemical detection reports, and dual detection improved peak identification in the urine samples. The proposed method was applied to the simultaneous determination of Catechol Estrogens in spiked urine in a preliminary study on Estrogens and PSA values in biopsy and prostate cancer patients.
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HPLC for stress‐free screening of potential prostate cancer marker Catechol Estrogens in urine using a diamond‐electrode electrochemical and a fluorescence detector
Journal of separation science, 2007Co-Authors: Masatoki Katayama, Kiyoshi Takamatsu, Satoru Kaneko, Keisuke Miyaji, Hiromichi Ishikawa, Yoshifumi MatsudaAbstract:Improvement of the sensitivity and specificity of a simultaneous stress-free screening method for Catechol Estrogens as a potential prostate cancer marker in urine has been accomplished by HPLC with a diamond-electrode electrochemical detector and a fluorescence detector. Since taking urine samples generates less stress (or pain) than the drawing of blood, the method can readily be applied to almost any patient, and will also assist in improving the sensitivity and specificity of the prostatic specific antigen test. Catechol Estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestriol) and Estrogens (estrone, estradiol, estriol) were separated on an Inertsil ODS-II column with acetonitrile-potassium dihydrogen phosphate (pH 3.0). The diamond-electrode electrochemical detector used had the great advantage of being a maintenance-free system, and could sequentially analyze hundreds of samples. Fluorescence detection improved the sensitivity 10-500 times (e. g., the LOD of 2-hydroxyestriol was improved 250 times) compared to previous electrochemical detection reports, and dual detection improved peak identification in the urine samples. The proposed method was applied to the simultaneous determination of Catechol Estrogens in spiked urine in a preliminary study on Estrogens and PSA values in biopsy and prostate cancer patients.
Hueichen Liang - One of the best experts on this subject based on the ideXlab platform.
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in situ click reaction coupled with quantitative proteomics for identifying protein targets of Catechol Estrogens
Journal of Proteome Research, 2018Co-Authors: Hueichen Liang, Yichen Liu, Hsin Chen, Chihyen Wang, Shun Fen Tzeng, Shuhui ChenAbstract:Catechol Estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography–mass spectrometry (LC–MS). A total of 334 liver proteins were repeatedly identified (n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific b...
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In Situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens
2018Co-Authors: Hueichen Liang, Yichen Liu, Hsin Chen, Chihyen Wang, Shun Fen Tzeng, Shuhui ChenAbstract:Catechol Estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography–mass spectrometry (LC–MS). A total of 334 liver proteins were repeatedly identified (n ≥ 2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 nonspecific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9), which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, Catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems
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site specific covalent modifications of human insulin by Catechol Estrogens reactivity and induced structural and functional changes
Scientific Reports, 2016Co-Authors: Chieh Ming Fang, Hueichen Liang, Juei Tang Cheng, Tzufan Wang, Chiao Chen Chen, Junghsiang Tai, Shuhui ChenAbstract:Proteins, covalently modified by Catechol Estrogens (CEs), were identified recently from the blood serum of diabetic patients and referred to as estrogenized proteins. Estrogenization of circulating insulin may occur and affect its molecular functioning. Here, the chemical reactivity of CEs towards specific amino acid residues of proteins and the structural and functional changes induced by the estrogenization of insulin were studied using cyclic voltammetry, liquid chromatography-mass spectrometry, circular dichroism spectroscopy, molecular modeling, and bioassays. Our results indicate that CEs, namely, 2- and 4-hydroxyl Estrogens, were thermodynamically and kinetically more reactive than the Catechol moiety. Upon co-incubation, intact insulin formed a substantial number of adducts with one or multiple CEs via covalent conjugation at its Cys 7 in the A or B chain, as well as at His10 or Lys29 in the B chain. Such conjugation was coupled with the cleavage of inter-chain disulfide linkages. Estrogenization on these sites may block the receptor-binding pockets of insulin. Insulin signaling and glucose uptake levels were lower in MCF-7 cells treated with modified insulin than in cells treated with native insulin. Taken together, our findings demonstrate that insulin molecules are susceptible to active estrogenization, and that such modification may alter the action of insulin.
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identification of endogenous site specific covalent binding of Catechol Estrogens to serum proteins in human blood
Toxicological Sciences, 2015Co-Authors: Chieh Ming Fang, Hueichen Liang, Tzufan Wang, Che Kai Chang, Shuhui ChenAbstract:Protein adducts covalently modified by Catechol Estrogens (CEs), referred to as estrogenized proteins, are potential biomarkers for estrogen homeostasis or exposure to environmental toxicants. However, serum proteins endogenously modified by CEs and the modification sites remain elusive. In this study, liquid chromatography-mass spectrometry (LC-MS)-based shotgun proteomics is applied to identify site-specific protein estrogenization in human blood via a systematic approach and stringent validation. We showed CEs, namely 2- and 4-hydroxyl Estrogens which are regarded as biomarkers for estrogen homeostasis, form covalent bonds with proteins, mainly via side chain Cys, Lys, or His residue. Estrogenization of purified human serum albumin (HSA) and immunoglobulin G (IgG) at specific sites was achieved by co-incubation and used as the standards to confirm the identified estrogenization in serum proteins. Based on a database search, estrogenized peptides derived from serum proteins in patient blood were identified; endogenous estrogenization of HSA and IgG-1 at multiple sites were confirmed as compared to the standards. Based on a test using Ellman's reagent, estrogenization produced stable products and irreversibly abolished the reactivity of Cys34-HSA, which is the most important antioxidant and nitric oxide carrier in blood. Given the importance of estrogen metabolism in environmental toxicology, further exploration of estrogenized proteins is warranted for biomarker discovery and/or new mechanisms in disease process.
Joachim G. Liehr - One of the best experts on this subject based on the ideXlab platform.
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Induction of Uterine Adenocarcinoma in CD-1 Mice by Catechol Estrogens
Cancer research, 2000Co-Authors: Retha R. Newbold, Joachim G. LiehrAbstract:Catechol Estrogens may mediate estrogen-induced carcinogenesis because 4-hydroxyestradiol induces DNA damage and renal tumors in hamsters, and this metabolite is formed in the kidney and estrogen target tissues by a specific estrogen 4-hydroxylase. We examined the carcinogenic potential of Catechol estrogen in an experimental model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal exposure to diethylstilbestrol. Outbred female CD-1 mice were treated with 2- or 4-hydroxyestradiol, 17beta-estradiol, or 17alpha-ethinyl estradiol on days 1-5 of neonatal life (2 microg/pup/day) and sacrificed at 12 or 18 months of age. Mice treated with 17beta-estradiol or 17a-ethinyl estradiol had a total uterine tumor incidence of 7% or 43%, respectively. 2-Hydroxyestradiol induced tumors in 12% of the mice, but 4-hydroxyestradiol was the most carcinogenic estrogen, with a 66% incidence of uterine adenocarcinoma. Both 2- and 4-hydroxylated Catechols were estrogenic and increased uterine wet weights in these neonates. These data demonstrate that both 2- and 4-hydroxyestradiol are carcinogenic metabolites. The high tumor incidence induced by 4-hydroxyestradiol supports the postulated role of this metabolite in hormone-associated cancers.
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microsome mediated 8 hydroxylation of guanine bases of dna by steroid Estrogens correlation of dna damage by free radicals with metabolic activation to quinones
Carcinogenesis, 1995Co-Authors: Xueliang Han, Joachim G. LiehrAbstract:Free radical generation by metabolic redox cycling between Catechol Estrogens and their quinones and subsequent hydroxyl radical damage to DNA have been proposed to mediate estrogen-induced renal carcinogenesis in the hamster. In this study the content of 8-hydroxy-2'-deoxy-guanosine (8-OHdG), a marker product of hydroxyl radical action, was examined in DNA incubated with a liver microsomal activating system and with Catechol Estrogens, equilenin-3,4-quinone or with parent Estrogens. Equilenin-3,4-quinone increased the formation of 8-OHdG by 50% over control levels. 4-Hydroxyestrone and 4-hydroxy-estradiol raised 8-OHdG contents significantly, to 1.61 +/- 0.79 and 1.27 +/- 0.31 8-OHdG/10(5) deoxyguanosine (dG) respectively over controls (0.68 +/- 0.25 8-OHdG/10(5) dG). The corresponding 2-hydroxylated Estrogens and the parent hormones estrone, estradiol and equilenin did not affect 8-hydroxylation of guanine bases of DNA. In incubations of Catechol Estrogens with microsomes and cumene hydroperoxide the 4-hydroxyEstrogens were oxidized to quinones more rapidly than the 2-hydroxyEstrogens. Our data support a mechanism of hydroxyl radical generation from Estrogens by redox cycling between 4-hydroxylated metabolites and their quinones. The rapid oxidation of 4-hydroxylated Estrogens to quinones, their redox cycling and hydroxyl radical damage to DNA is consistent with the previously reported carcinogenic activities of 4-hydroxylated, but not of 2-hydroxylated, Catechol Estrogens.
Estelle Rathahao - One of the best experts on this subject based on the ideXlab platform.
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liquid chromatography coupled to negative electrospray ion trap mass spectrometry for the identification of isomeric glutathione conjugates of Catechol Estrogens
International Journal of Mass Spectrometry, 2004Co-Authors: Estelle Rathahao, Isabelle Jouanin, A Page, A. Paris, Laurent DebrauwerAbstract:Conjugation to glutathione (GSH) represents an important detoxification pathway for preventing DNA damage due to oxidation products of Catechol Estrogens. In order to identify isomeric GSH conjugates of Catechol Estrogens, liquid chromatography coupled to electrospray/ion trap mass spectrometry was used. For this purpose, both positive and negative ion modes were applied, generating protonated and deprotonated species, respectively. Energy-resolved fragmentation of each isomeric quasi-molecular ion was achieved in two regions of the mass spectrometer: (i) the mass analyzer (ion trap mass spectrometer) and (ii) the interface region of the electrospray ionization source. The resonance excitation of [M+H]+ protonated ions carried out into the ion trap did not show any isomeric differentiation. Although ion source fragmentation of these same species allowed identification of each isomer, this method requires a good chromatographic separation, making it inefficient for the analysis of low sample amounts from in vitro or in vivo sources. Conversely, using resonance excitation of deprotonated ions, isomer distinction could be achieved. Thus, this technique should yield the best data for the direct characterization of isomers of Catechol estrogen–GSH conjugates from biological samples.
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Liquid chromatography coupled to negative electrospray/ion trap mass spectrometry for the identification of isomeric glutathione conjugates of Catechol Estrogens
International Journal of Mass Spectrometry, 2004Co-Authors: Estelle Rathahao, Isabelle Jouanin, A Page, A. Paris, Laurent DebrauwerAbstract:Conjugation to glutathione (GSH) represents an important detoxification pathway for preventing DNA damage due to oxidation products of Catechol Estrogens. In order to identify isomeric GSH conjugates of Catechol Estrogens, liquid chromatography coupled to electrospray/ion trap mass spectrometry was used. For this purpose, both positive and negative ion modes were applied, generating protonated and deprotonated species, respectively. Energy-resolved fragmentation of each isomeric quasi-molecular ion was achieved in two regions of the mass spectrometer: (i) the mass analyzer (ion trap mass spectrometer) and (ii) the interface region of the electrospray ionization source. The resonance excitation of [M+H]+ protonated ions carried out into the ion trap did not show any isomeric differentiation. Although ion source fragmentation of these same species allowed identification of each isomer, this method requires a good chromatographic separation, making it inefficient for the analysis of low sample amounts from in vitro or in vivo sources. Conversely, using resonance excitation of deprotonated ions, isomer distinction could be achieved. Thus, this technique should yield the best data for the direct characterization of isomers of Catechol estrogen–GSH conjugates from biological samples.
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Adduction of Catechol Estrogens to nucleosides.
Steroids, 2002Co-Authors: Isabelle Jouanin, Laurent Debrauwer, Gwénola Fauglas, Alain Paris, Estelle RathahaoAbstract:Abstract We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the Catechol Estrogens (CE) of estrone, estradiol-17β and estradiol-17α. The crude products are obtained in a one-pot synthesis through oxidation of Catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium. In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MSn). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-Catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for α and β isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS2 and MS3 experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety.
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Adduction of Catechol Estrogens to nucleosides.
Steroids, 2002Co-Authors: Isabelle Jouanin, Laurent Debrauwer, Gwénola Fauglas, Alain Paris, Estelle RathahaoAbstract:We report the formation, detection, quantitation and structural characterization of products resulting from the adduction of deoxynucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine and 5-methyldeoxycytidine) to the Catechol Estrogens (CE) of estrone, estradiol-17beta and estradiol-17 alpha. The crude products are obtained in a one-pot synthesis through oxidation of Catechols to quinones and subsequent Michael-type reaction with the deoxynucleosides in acidic medium. In all experiments, adducts are detected by electrospray ionization mass spectrometry analysis after HPLC separation (LC/ESI/MS(n)). The two pyrimidines deoxycytidine and 5-methyldeoxycytidine yield only CE adducts to deoxynucleosides, which correspond to stable adducts on DNA. For purines, the results depend on the CE (2,3- or 3,4-Catechols) used, the function and configuration on carbon 17 (ketone for estrone, alcohol for alpha and beta isomers of estradiol), and on the purine itself (deoxyadenosine or deoxyguanosine). Both stable adducts and deglycosylated adducts are formed, and therefore formation of stable adducts on DNA as well as the loss of purines from the DNA strands could be possible. MS(2) and MS(3) experiments prove to be relevant for further structural determinations, enabling in some cases the elucidation of the regiochemistry of adduction on the A and B rings of the steroid moiety.