74 results on '"Hauh-Jyun Candy Chen"'
Search Results
2. Response to 'Malondialdehyde-Induced Post-Translational Modification of Human Hemoglobin'
- Author
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Hauh-Jyun Candy Chen and Yan-Ling Liao
- Subjects
General Chemistry ,Biochemistry - Published
- 2023
3. Malondialdehyde-Induced Post-translational Modifications in Hemoglobin of Smokers by NanoLC-NSI/MS/MS Analysis
- Author
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Hauh-Jyun Candy Chen, Chau-Yi Chen, Ya-Hsuan Fang, Kai-Wei Hung, and Deng-Chyang Wu
- Subjects
Hemoglobins ,Smokers ,Tandem Mass Spectrometry ,Lysine ,Malondialdehyde ,Humans ,General Chemistry ,Biochemistry ,Protein Processing, Post-Translational ,Biomarkers - Abstract
Malondialdehyde (MDA) is the most abundant α,β-unsaturated aldehyde generated from endogenous peroxidation of polyunsaturated fatty acids and is present in cigarette smoke. Post-translational modifications of blood hemoglobin can serve as biomarkers for exposure to chemicals. In this study, two types of MDA-induced modifications, the
- Published
- 2022
4. Characterization and Quantification of Acrolein-Induced Modifications in Hemoglobin by Mass Spectrometry─Effect of Cigarette Smoking
- Author
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Hauh-Jyun Candy Chen, Shu-Wei Cheng, Nai-Ying Chen, and Deng-Chyang Wu
- Subjects
Hemoglobins ,Tobacco ,Humans ,Mouth Neoplasms ,General Medicine ,Acrolein ,Toxicology ,Peptides ,Mass Spectrometry ,Cigarette Smoking - Abstract
Exposure to acrolein, the smallest α, β-unsaturated aldehyde, in humans originates from cigarette smoking and other environmental sources, food cooking, and endogenous lipid peroxidation and metabolism. The protein modification caused by acrolein is associated with various diseases, including cancer, cardiovascular, and neurodegenerative diseases. In this study, acrolein-modified human hemoglobin was reduced by sodium borohydride. Thus, three types of modifications, that is, Schiff base, Michael addition, and formyl-dehydropiperidion adducts, were converted to the corresponding stable adducts, leading to mass increases of 40.0313, 58.0419, and 96.0575 Da, respectively. These stable acrolein-modified hemoglobin peptides were identified by nanoflow liquid chromatography coupled to a high-resolution nanoelectrospray ionization tandem mass spectrometry. Among the 26 different types and sites of modifications, 15 of them showed a dose-dependent increase with increasing concentrations of acrolein. To investigate the role of acrolein-induced modifications in smoking and oral cancer, the 15 dose-responsive acrolein-modified peptides, together with three ethylated peptides previously identified, were quantified in oral cancer patients, healthy smokers, and healthy nonsmokers. The results reveal that the relative extents of the Michael-type adduct at α-Lys-16, α-His-50, and β-Lys-59 are significantly higher in smokers (oral cancer and healthy) than in nonsmokers. Areas under the receiver operating characteristic curve of these peptides range from 0.7500 to 0.9375, indicating the ability to discriminate smokers from nonsmokers. Additionally, these acrolein-modified peptides correlate with three ethylated peptides at the
- Published
- 2022
5. Quantitation of Nitration, Chlorination, and Oxidation in Hemoglobin of Breast Cancer Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry
- Author
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Chi-Wen Tu, Kuan-Ching Liao, and Hauh-Jyun Candy Chen
- Subjects
Halogenation ,Breast Neoplasms ,010501 environmental sciences ,Toxicology ,Tandem mass spectrometry ,01 natural sciences ,Hemoglobins ,03 medical and health sciences ,Blood serum ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Humans ,Nanotechnology ,030304 developmental biology ,0105 earth and related environmental sciences ,0303 health sciences ,Chromatography ,Chemistry ,Selected reaction monitoring ,Nitrosylation ,Albumin ,General Medicine ,Middle Aged ,Blood proteins ,Female ,Hemoglobin ,Oxidation-Reduction ,Chromatography, Liquid - Abstract
Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p 0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.
- Published
- 2021
6. Multiple oxidative and advanced oxidative modifications of hemoglobin in gastric cancer patients measured by nanoflow LC-MS/MS
- Author
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Deng-Chyang Wu, Tzu-Chun Yang, Shun-Xiang Hu, and Hauh-Jyun Candy Chen
- Subjects
Aspartic Acid ,Biochemistry (medical) ,Clinical Biochemistry ,General Medicine ,Biochemistry ,Hemoglobins ,Oxidative Stress ,Stomach Neoplasms ,Tandem Mass Spectrometry ,Humans ,Tyrosine ,Histidine ,Peptides ,Biomarkers ,Chromatography, Liquid - Abstract
Overproduction of the reactive oxygen, nitrogen, and chlorine species by the immune systems during chronic infection and inflammation can cause structural and functional changes of cellular proteins. The high abundance of hemoglobin in blood makes hemoglobin adducts suitable biomarkers for assessing the damage of these reactive species in the body. In this study, a total of 23 types and sites of modification in human hemoglobin were simultaneously analyzed, including monooxygenation of histidine, tyrosine, methionine, and aspartate, conversion of histidine to aspartate and hydroxyaspartate, as well as chlorination and nitration of tyrosine residues. Hemoglobin was isolated from the blood of the study subjects, digested into peptides, and the extents of these modifications were quantified relative to their parent peptides using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC - NSI-MS/MS-SRM). The extents of monooxygenation at β-His-77 and β-Tyr-130, chlorination at α-Tyr-24 and β-Tyr-130, and nitrosylation at α-Tyr-24 were elevated in gastric cancer patients. Conversely, conversion of histidine to aspartate at α-His-20, α-His-50, β-His-2, β-His-143, and monooxygenation at β-His-143 were decreased in gastric cancer patients. The areas under the receiver operating characteristics (AUC of ROC) curve of these ten types and sites of hemoglobin modifications were between 0.7644 and 0.9644. The ratio of conversion of histidine to hydroxyaspartate versus conversion of histidine to aspartate was significantly higher in gastric cancer patients at α-His-20, α-His-50, and β-His-143 (p 0.05) with AUC of ROC ranging between 0.7689 and 0.9178. To our knowledge, this is the first report of simultaneous measurement of multiple types of oxidative and advanced oxidative hemoglobin modifications in gastric cancer patients. The results revealed elevated levels of oxidative stress-induced protein damage in gastric cancer patients and the potential of using these modifications of hemoglobin as biomarkers for evaluation of oxidative stress in one drop of blood.
- Published
- 2022
7. Analysis of Oxidative and Advanced Oxidative Modifications in Hemoglobin of Oral Cancer Patients by Mass Spectrometry
- Author
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Nai-Ying Chen, Yu-Sheng Lin, Deng-Chyang Wu, Hauh-Jyun Candy Chen, Chin-Wei Wu, and Tsai-Shiuan Lin
- Subjects
Cellular respiration ,Oxidative phosphorylation ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Mass Spectrometry ,Analytical Chemistry ,Hemoglobins ,medicine ,Humans ,Globin ,Chromatography, High Pressure Liquid ,Histidine ,chemistry.chemical_classification ,Reactive oxygen species ,010401 analytical chemistry ,Cancer ,medicine.disease ,0104 chemical sciences ,chemistry ,Biochemistry ,Mouth Neoplasms ,Hemoglobin ,Reactive Oxygen Species ,Oxidation-Reduction ,Oxidative stress - Abstract
We are exposed to endogenous reactive oxygen species (ROS) produced during normal aerobic metabolism and by the innate immune systems. Excessive production of ROS is critically important in disease onset and progression. Post-translational oxidative modifications of hemoglobin have been used as a surrogate biomarker for monitoring the oxidative stress in vivo. In this study, the Fenton reaction was used as a model to produce ROS and react with human hemoglobin. After trypsin digestion, the types and sites of modifications were characterized by a nanoflow LC-nanoelectrospray ionization high-resolution mass spectrometer. Besides oxidation at certain sites of Met, His, and Tyr, conversion of histidine to aspartate and hydroxyaspartate was identified at four sites. Furthermore, advanced oxidation of histidine to hydroxyaspartate was identified at two sites. Elevated oxidative stress is tightly associated with oral cancer. The relative extent of modification at the identified sites was quantified relative to the native peptide present in the digest as the reference peptide in hemoglobin from 18 oral cancer patients and 15 healthy control subjects. The results revealed that the extents of oxidation at β-His-77 and β-Asp-99 of globin were significantly elevated in oral cancer patients compared to healthy subjects, while the extents of conversion of histidine residues at α-His-20, α-His-50, and β-His-2 to aspartate were significantly decreased. Furthermore, the advanced oxidation of histidine to hydroxyaspartate at α-His-50 and β-His-2 is also significantly higher in oral cancer patients than in healthy subjects (p < 0.05). To our knowledge, this advanced oxidation of histidine to hydroxyaspartate is a new post-translational protein modification, and it is found in human hemoglobin isolated from blood. This advanced oxidative modification in hemoglobin might be a potential biomarker to assess oxidative stress in oral cancer patients.
- Published
- 2019
8. Stability of glyoxal- and methylglyoxal-modified hemoglobin on dried blood spot cards as analyzed by nanoflow liquid chromatography tandem mass spectrometry
- Author
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Hauh-Jyun Candy Chen and Yi-Chun Teng
- Subjects
030309 nutrition & dietetics ,lcsh:TX341-641 ,Tandem mass spectrometry ,01 natural sciences ,Hemoglobins ,03 medical and health sciences ,symbols.namesake ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Humans ,Nanotechnology ,Pharmacology ,0303 health sciences ,Chromatography ,Protein Stability ,010401 analytical chemistry ,Methylglyoxal ,lcsh:RM1-950 ,Glyoxal ,0104 chemical sciences ,Dried blood spot ,Maillard reaction ,lcsh:Therapeutics. Pharmacology ,chemistry ,symbols ,Dried Blood Spot Testing ,Hemoglobin ,lcsh:Nutrition. Foods and food supply ,Chromatography, Liquid ,Food Science ,Blood sampling - Abstract
Blood sampling by the dried blood spot (DBS) technique has become commonly applied in newborn screening. It is often used for analysis of small molecules, such as metabolites. Recently, DBS sampling has been applied for quantification of post-translational protein modifications. Glyoxal and methylglyoxal are two simple oxoaldehydes released from glycated proteins in the Maillard reaction. They are widely distributed in the environment (e.g. cigarette smoke) and found in foods and beverages. Glyoxal and methylglyoxal are shown to react with biomolecules including DNA and proteins. In this laboratory, we previously identified the sites of modification by these two oxoaldehydes in human hemoglobin and found that the extents of modification at certain sites of lysine and arginine residues are significantly higher in type 2 diabetes mellitus patients than in nondiabetic individuals. In this study, we examine the stability of these modifications of hemoglobin stored on DBS cards at room temperature or 4 °C in the ambient air. After hemoglobin was extracted from the DBS cards, it was digested by trypsin and analyzed by nanoflow liquid chromatography coupled with nanospray ionization tandem mass spectrometry. The results show that the extents of all these PTMs are stable within 14 and 21 days when stored on DBS at room temperature and at 4 °C, respectively. Extraction of globin from DBS cards is mostly advantageous for hemolytic blood samples. This assay is sensitive as only a quarter of a DBS card containing ca. 12 μL of blood is required. Thus, it is practically useful to measure the extents of glyoxal- and methylglyoxal-induced hemoglobin modifications from DBS cards. Keywords: Blood, Dried blood spot, Glyoxal, Hemoglobin, Mass spectrometry, Methylglyoxal, Post-translational modifications
- Published
- 2019
9. Correlation between Glyoxal-Induced DNA Cross-Links and Hemoglobin Modifications in Human Blood Measured by Mass Spectrometry
- Author
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Chun-Ting Liu, Yi-Jou Li, and Hauh-Jyun Candy Chen
- Subjects
DNA damage ,010501 environmental sciences ,Toxicology ,Tandem mass spectrometry ,01 natural sciences ,Lipid peroxidation ,Hemoglobins ,03 medical and health sciences ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,In vivo ,Humans ,Globin ,030304 developmental biology ,0105 earth and related environmental sciences ,0303 health sciences ,Molecular Structure ,Chemistry ,DNA ,Glyoxal ,General Medicine ,Cross-Linking Reagents ,Biochemistry ,Hemoglobin ,Chromatography, Liquid - Abstract
Glyoxal is an oxoaldehyde generated from the degradation of glucose-protein conjugates and from lipid peroxidation in foods and in vivo, and it is also present in the environment (e.g., cigarette smoke). The major endogenous source of glyoxal is glucose autoxidation, and the glyoxal concentrations in plasma are higher in diabetic patients than in nondiabetics. Glyoxal reacts with biomolecules forming covalently modified DNA and protein adducts. We previously developed sensitive and specific assays based on nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) for quantification of DNA cross-linked adducts (dG-gx-dC and dG-gx-dA) and for hemoglobin adducts derived from glyoxal. In this study, we isolated and analyzed both leukocyte DNA and hemoglobin from the blood of diabetic patients and compared the adduct levels with those from nondiabetic subjects using the modified assays. The results indicated that the extents of glyoxal-induced hemoglobin modifications on α-Lys-11, α-Arg-92, β-Lys-17, and β-Lys-66 were statistically higher in diabetic patients than nondiabetics and they correlated with HbA1c significantly. Moreover, the levels of dG-gx-dC in leukocyte DNA correlated positively with the extents of globin modification at α-Lys-11 and β-Lys-17, while levels of dG-gx-dA correlated with those at α-Lys-11 and α-Arg-92 in nonsmoking subjects. Comparing the levels and the correlation coefficients of these hemoglobin and DNA adducts including or excluding smokers, it appears that smoking is not a major contributor to glyoxal-induced adduction of hemoglobin and leukocyte DNA. To the best of our knowledge, this is one of the few reports of positive correlation between DNA and protein adducts of the same compound (glyoxal) in the blood from the same subjects. Because of the high abundance of hemoglobin in blood, the results indicate that quantification of glyoxal-modified peptides in hemoglobin might serve as a dosimetry for glyoxal and a practical surrogate biomarker for assessing glyoxal-induced DNA damage and its prevention.
- Published
- 2018
10. Characterization of DNA-protein cross-links induced by oxanine: Cellular damage derived from nitric oxide and nitrous acid
- Author
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Hauh-Jyun Candy Chen, Chia-Jong Hsieh, Li-Ching Shen, and Chia-Ming Chang
- Subjects
Thiols -- Chemical properties ,Nuclear magnetic resonance -- Research ,Proteins -- Crosslinking ,Proteins -- Research ,Biological sciences ,Chemistry - Abstract
Thioester and the amine adduct, the two reaction products of oxanine were characterized, considering their UV, NMR and mass spectra. The findings suggested that the thiol group of the amino acid side chain is reactive toward oxanine.
- Published
- 2007
11. Age-Associated Methylation in Human Hemoglobin and Its Stability on Dried Blood Spots As Analyzed by Nanoflow Liquid Chromatography Tandem Mass Spectrometry
- Author
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Sun Wai Ip and Hauh-Jyun Candy Chen
- Subjects
Adult ,Male ,0301 basic medicine ,Toxicology ,Methylation ,Hemoglobins ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Humans ,Amino Acid Sequence ,Aged ,Dried Blood Spot Testing ,chemistry.chemical_classification ,Chromatography ,Spots ,Protein Stability ,Biomolecule ,Selected reaction monitoring ,General Medicine ,Middle Aged ,Dried blood spot ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Female ,Hemoglobin ,Peptides ,Chromatography, Liquid - Abstract
Methylation of biomolecules is involved in many important biological processes. The contributing methylating agents arise from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as a molecular dosimeter for monitoring chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin by nanoflow liquid chromatography tandem mass spectrometry under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in hemoglobin were quantified in nonsmoking subjects at various ages in this study. Among the nine methylation sites, we found that the extents of methylation were significantly higher in elderly subjects at the N-terminal and His-20 of α-globin, and at the N-terminal and Glu-26 of β-globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. We also examined the stability of methylated and ethylated hemoglobin when stored on dried blood spot cards. The extents of these modifications on hemoglobin are stable for at least 4 weeks stored at room temperature. Our results suggest that age should be considered as a factor when measuring hemoglobin methylation and that dried blood spot is a valuable biomonitoring technique for hemoglobin modifications in epidemiological studies.
- Published
- 2018
12. A Stable Isotope Dilution Nanoflow Liquid Chromatography Tandem Mass Spectrometry Assay for the Simultaneous Detection and Quantification of Glyoxal-Induced DNA Cross-Linked Adducts in Leukocytes from Diabetic Patients
- Author
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Tsai-Shiuan Lin, Hauh-Jyun Candy Chen, Ya-Lang Chang, Chiung-Fong Hsiao, and Yi-Chun Teng
- Subjects
0301 basic medicine ,Indicator Dilution Techniques ,Tandem mass spectrometry ,Analytical Chemistry ,Adduct ,DNA Adducts ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Leukocytes ,Humans ,Chromatography ,Molecular Structure ,Chemistry ,Stable isotope ratio ,Selected reaction monitoring ,Glyoxal ,Triple quadrupole mass spectrometer ,030104 developmental biology ,Diabetes Mellitus, Type 2 ,030220 oncology & carcinogenesis ,Nucleic acid ,Chromatography, Liquid - Abstract
Glyoxal (gx) is a bifunctional electrophile capable of cross-linking DNA. Although it is present in foods and from the environment, endogenous formation of glyoxal occurs through metabolism of carbohydrates and oxidation of lipids and nucleic acids. Plasma concentrations of glyoxal are elevated in in diabetes mellitus patients compared to nondiabetics. The most abundant 2′-deoxyribonucleoside adducts cross-linked by glyoxal are dG-gx-dC, dG-gx-dA, and dG-gx-dG. These DNA cross-links can be mutagenic by damaging the integrity of the DNA structure. Herein, we developed a highly sensitive and specific assay for the simultaneous detection and quantification of the dG-gx-dC and dG-gx-dA cross-links based on stable isotope dilution (SID) nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selected reaction monitoring mode and using a triple quadrupole mass spectrometer. The entire assay procedure involved addition of the stable isotope standards [15N5...
- Published
- 2017
13. Simultaneous Mass Spectrometric Analysis of Methylated and Ethylated Peptides in Human Hemoglobin: Correlation with Cigarette Smoking
- Author
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Fu-Di Lin, Hauh-Jyun Candy Chen, and Sun Wai Ip
- Subjects
Adult ,Male ,0301 basic medicine ,Alkylating Agents ,Alkylation ,Ethyl methanesulfonate ,Toxicology ,Mass spectrometry ,Methylation ,01 natural sciences ,Cigarette Smoking ,Hemoglobins ,Young Adult ,03 medical and health sciences ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Protein methylation ,Humans ,Amino Acid Sequence ,Chromatography ,Chemistry ,010401 analytical chemistry ,Selected reaction monitoring ,General Medicine ,Mass spectrometric ,0104 chemical sciences ,Methyl methanesulfonate ,030104 developmental biology ,Biochemistry ,Female ,Hemoglobin ,Peptides - Abstract
Alkylating agents contained in cigarettes smoke might be related to cancer development. Post-translational protein methylation and ethylation may cause alteration of protein functions. Human hemoglobin (Hb) has been a target for molecular dosimetry because of its easy accessibility. The goal of this study is to investigate the relationship between the levels of methylation and ethylation at specific sites of Hb with smoking. Because of the low extent of modification of Hb isolated from blood, the methylation and ethylation sites were identified in Hb incubated with a methylating agent (methyl methanesulfonate, MMS) and ethylating agent (ethyl methanesulfonate, EMS), respectively, by accurate mass measurements. After trypsin digestion, the modification sites were identified by nanoflow LC-nanospray ionization coupled with high-resolution mass spectrometry. The selected reaction monitoring mode was used to quantify the relative extent of methylation and ethylation in human Hb incubated with MMS and EMS, respectively. Methylation occurred at 9 sites, including
- Published
- 2017
14. Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry
- Author
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Chih-Huang Fan, Hauh-Jyun Candy Chen, and Ya-Fen Yang
- Subjects
0301 basic medicine ,Bioanalysis ,chemistry.chemical_element ,Toxicology ,Tandem mass spectrometry ,01 natural sciences ,Oxygen ,Hemoglobins ,03 medical and health sciences ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,Humans ,Globin ,Chromatography ,Chemistry ,010401 analytical chemistry ,General Medicine ,Reactive Nitrogen Species ,0104 chemical sciences ,Dried blood spot ,Blood ,030104 developmental biology ,Biochemistry ,Hemoglobin ,Sample collection ,Reactive Oxygen Species ,Chromatography, Liquid - Abstract
Dried blood spot (DBS) is an emerging microsampling technique for the bioanalysis of small molecules, including fatty acids, metabolites, drugs, and toxicants. DBS offers many advantages as a sample format including easy sample collection and cheap sample shipment. Hemoglobin adducts have been recognized as a suitable biomarker for monitoring chemical exposure. We previously reported that certain modified peptides in hemoglobin derived from reactive chlorine, nitrogen, and oxygen species are associated with factors including smoking, diabetes mellitus, and aging. However, the stability of these oxidation-induced modifications of hemoglobin remains unknown and whether they can be formed artifactually during storage of DBS. To answer these questions, globin extracted from the DBS cards was analyzed, and the stability of the modifications was evaluated. After storage of the DBS cards at 4 °C or room temperature up to 7 weeks, we isolated globin from a quarter of the spot every week. The extents of 11 sites and types of post-translational modifications (PTMs), including nitration and nitrosylation of tyrosine and oxidation of cysteine and methionine residues, in human hemoglobin were measured in the trypsin digest by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) using selected reaction monitoring. The extents of all these PTMs are stable within 14 days when stored on DBS at room temperature and at 4 °C, while those from direct extraction of fresh blood are stable for at least 8 weeks when stored as an aqueous solution at -20 °C. Extraction of globin from a DBS card is of particular importance for hemolytic blood samples. To our knowledge, this is the first report on the stability of oxidative modifications of hemoglobin on DBSs, which are stable for 14 days under ambient conditions (room temperature, in air). Therefore, it is feasible and convenient to analyze these hemoglobin modifications from DBSs in studies involving large populations.
- Published
- 2016
15. Analysis of cysteine glutathionylation in hemoglobin of gastric cancer patients using nanoflow liquid chromatography/triple-stage mass spectrometry
- Author
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Pang-Yen Lai, Deng-Chyang Wu, and Hauh-Jyun Candy Chen
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,Peptide ,beta-Globins ,Protein glutathionylation ,medicine.disease_cause ,01 natural sciences ,Analytical Chemistry ,chemistry.chemical_compound ,Young Adult ,alpha-Globins ,Stomach Neoplasms ,medicine ,Humans ,Cysteine ,Spectroscopy ,Aged ,chemistry.chemical_classification ,Aged, 80 and over ,Chromatography ,010401 analytical chemistry ,Organic Chemistry ,Selected reaction monitoring ,Glutathione ,Middle Aged ,Trypsin ,0104 chemical sciences ,chemistry ,Female ,Hemoglobin ,Oxidation-Reduction ,Oxidative stress ,medicine.drug ,Chromatography, Liquid - Abstract
Glutathione is an intracellular antioxidant capable of scavenging free radicals and detoxifying electrophiles from endogenous and exogenous sources via the free thiol group. Post-translational glutathionylation at cysteine residues of proteins can affect the structure and cause a functional change of proteins. Protein glutathionylation has been proven to reflect the cellular redox status. Our previous report indicates that the levels of glutathionylation in hemoglobin from peripheral blood of smokers are significantly higher than in nonsmokers. In this study, a nanoflow liquid chromatography/nanospray ionization triple-stage mass spectrometric (nanoLC/NSI-MS3 ) method with a linear ion trap mass spectrometer was employed to quantify glutathionylated peptides in the trypsin digests of hemoglobin from gastric cancer patients. We compare the extent of glutathionylation in hemoglobin from nonsmoking gastric cancer patients with that from nonsmoking healthy adults. Using a carboxymethylated peptide as the reference peptide, the relative quantification of each glutathionylated peptide was measured as the peak area ratio of the modified peptide versus the sum of the peak areas of the modified and the carboxymethylated parent peptide in the selected reaction monitoring chromatograms. Using this method, we found that the extents of glutathionylation at Cys-104 of the α-globin and Cys-93 of β-globulin hemoglobin from 10 gastric cancer patients were significantly higher than those from 14 normal individuals with p values
- Published
- 2019
16. Mass Spectrometric Analysis of Glyoxal and Methylglyoxal-Induced Modifications in Human Hemoglobin from Poorly Controlled Type 2 Diabetes Mellitus Patients
- Author
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Pin-Fan Chen, Yu-Chin Chen, Chiung-Fong Hsiao, and Hauh-Jyun Candy Chen
- Subjects
Male ,Spectrometry, Mass, Electrospray Ionization ,Arginine ,Lysine ,Peptide ,Toxicology ,Tandem mass spectrometry ,Hemoglobins ,chemistry.chemical_compound ,Humans ,chemistry.chemical_classification ,Chromatography ,Smoking ,Methylglyoxal ,Selected reaction monitoring ,Glyoxal ,General Medicine ,Middle Aged ,Reference Standards ,Pyruvaldehyde ,Diabetes Mellitus, Type 2 ,chemistry ,Biochemistry ,Female ,Hemoglobin ,Peptides - Abstract
Glyoxal and methylglyoxal are oxoaldehydes derived from the degradation of glucose-protein conjugates and from lipid peroxidation, and they are also present in the environment. This study investigated the site-specific reaction of glyoxal and methylglyoxal with the amino acid residues on human hemoglobin using a shot-gun proteomic approach with nanoflow liquid chromatography/nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). In human hemoglobin incubated with glyoxal, modification on 8 different sites, including lysine residues at α-Lys-11, α-Lys-16, α-Lys-56, β-Lys-17, β-Lys-66, β-Lys-144, and arginine residues at α-Arg-92 and β-Arg-30, was observed using a data-dependent scan. In methylglyoxal-treated hemoglobin, there were specific residues, namely, α-Arg-92, β-Lys-66, β-Arg-30, and β-Lys-144, forming carboxyethylation as well as the dehydrated product hydroimidazolone at α-Arg-92 and β-Arg-30. These lysine and arginine modifications were confirmed by accurate mass measurement and the MS(2) and MS(3) spectra. The most intensive signal of each modified peptide was used as the precursor ion to perform the product ion scan. The relative extent of modifications was semiquantified simultaneously relative to the native reference peptide by nanoLC-NSI/MS/MS under the selected reaction monitoring (SRM) mode. The extent of these modifications increased dose-dependently with increasing concentrations of glyoxal or methylglyoxal. Six out of the eight modifications induced by glyoxal and three out of the six modifications induced by methylglyoxal were detected in hemoglobin freshly isolated from human blood samples. The relative extent of modification of these post-translational modifications was quantified in poorly controlled type 2 diabetes mellitus patients (n = 20) and in nondiabetic control subjects (n = 21). The results show that the carboxymethylated peptides at α-Lys-16, α-Arg-92, β-Lys-17, β-Lys-66, and the peptide at α-Arg-92 with methylglyoxal-derived hydroimidazolone are significantly higher in diabetic patients than in normal individuals (p value
- Published
- 2015
17. Multistage Mass Spectrometric Analysis of Human Hemoglobin Glutathionylation: Correlation with Cigarette Smoking
- Author
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Hauh-Jyun Candy Chen, Wen-Peng Lin, Shei-Da Chiu, and Chih-Huang Fan
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,Adolescent ,Molecular Sequence Data ,Toxicology ,Protein glutathionylation ,medicine.disease_cause ,Mass Spectrometry ,Hemoglobins ,Young Adult ,chemistry.chemical_compound ,Protein structure ,medicine ,Humans ,Amino Acid Sequence ,Cysteine ,chemistry.chemical_classification ,Glutathione Disulfide ,Smoking ,General Medicine ,Glutathione ,Trypsin ,chemistry ,Biochemistry ,Thiol ,Female ,Hemoglobin ,Peptides ,Oxidative stress ,medicine.drug - Abstract
Protein glutathionylation is an important protein post-translational modification associated with oxidative stress, in which the thiol groups of cysteine residues react with glutathione and form disulfide bonds. Glutathionylation has been shown to affect protein structure and modulate protein function, and is implicated in the regulation of signaling and metabolic pathways. Glutathionylation of human hemoglobin has been known for decades. However, only glutathionylation on Cys-93 of β-globin has been characterized. In this study, we used nanoflow liquid chromatography-nanospray ionization multistage mass spectrometry (nanoLC-NSI/MS(n)) under a data-dependent scan mode to identify sites of glutathionylation in human hemoglobin by accurate mass measurement and by their MS(2) and MS(3) spectra. After human hemoglobin was incubated with oxidized glutathione, alkylated and trypsin digested, all three cysteine residues, i.e., α-Cys-104, β-Cys-93, and β-Cys-112, were found to be glutathionylated. Glutathionylation at these sites was also detected in hemoglobin freshly isolated from human blood samples by the consecutive reaction monitoring at the MS(3) scan stage, and the extent of modification of each site was quantified relative to the alkylated parent peptide in the selected reaction monitoring (SRM) chromatograms. The peak area ratio of glutathionylated and alkylated parent peptides under MS(3) and MS(2)-SRM, respectively, represents the relative extent of glutathionylation. The extents of glutathionylation at α-Cys-104 and β-Cys-93 in hemoglobin of 20 smokers were significantly higher than those in 20 nonsmokers with a p value of 0.0008 and0.0001, respectively. Moreover, there are statistically significant correlations between the extent of glutathionylation at α-Cys-104 and β-Cys-93 and the number of cigarettes smoked per day as well as smoking index. This assay is highly specific and sensitive as it requires as little as 2 μg of hemoglobin isolated from one drop of blood. These results suggest that this assay might be feasible in measuring the extent of glutathionylation in hemoglobin as a low-invasive biomarker of oxidative stress. To our knowledge, this is the first report identifying all three cysteine residues being glutathionylated in human hemoglobin and quantifying the extent of glutathionylation at the peptide level using multistage mass spectrometry.
- Published
- 2014
18. Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry
- Author
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Chin-Ron Lee and Hauh-Jyun Candy Chen
- Subjects
Adult ,Male ,Saliva ,Chromatography ,Chemistry ,Smoking ,Selected reaction monitoring ,General Medicine ,Urine ,Toxicology ,Mass spectrometry ,Tandem mass spectrometry ,DNA Adducts ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Liquid chromatography–mass spectrometry ,DNA adduct ,Humans ,DNA ,Chromatography, Liquid ,Thymidine - Abstract
Smoking cigarette increases levels of certain ethylated DNA adducts in certain tissues and urine. Cigarette smoking is a major risk factor of various cancers and DNA ethylation is involved in smoking-related carcinogenesis. Among the ethylated DNA adducts, O(2)-ethylthymidine (O(2)-edT) and the promutagenic O(4)-ethylthymidine (O(4)-edT) are poorly repaired and they can accumulate in vivo. Using an accurate, highly sensitive, and quantitative assay based on stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS), O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-edT adducts in human salivary DNA were simultaneous detected and quantified. Saliva is easily accessible and available and it can be a potential target in searching for noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 20 smokers and 13 nonsmokers were analyzed. Starting with 50 μg of DNA isolated from about 3.5 mL of saliva, levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' salivary DNA samples were 5.3±6.2, 4.5±5.7, 4.2±8.0 in 10(8) normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p0.0001) were observed between levels of O(2)-edT and N(3)-edT (γ=0.7388), between levels of O(2)-edT and O(4)-edT (γ=0.8839), and between levels of N(3)-edT, and O(4)-edT (γ=0.7835). To the best of our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA, which might be potential biomarkers for exposure to ethylating agents and possibly for cancer risk assessment.
- Published
- 2014
19. Simultaneous quantification of ethylpurine adducts in human urine by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry
- Author
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Chao-Ray Lin and Hauh-Jyun Candy Chen
- Subjects
Detection limit ,Carbon Isotopes ,Chromatography ,Nitrogen Isotopes ,Chemistry ,Solid Phase Extraction ,Organic Chemistry ,Extraction (chemistry) ,Indicator Dilution Techniques ,General Medicine ,Urine ,Mass spectrometry ,Tandem mass spectrometry ,Biochemistry ,Analytical Chemistry ,Adduct ,DNA Adducts ,Limit of Detection ,Purines ,Tandem Mass Spectrometry ,Ionization ,DNA adduct ,Humans ,Chromatography, Liquid - Abstract
Ethylating agents contained in cigarette smoke can damage DNA producing ethylated DNA adducts, including N(3)-ethyladenine (3-EtAde) and N(7)-ethylguanine (7-EtGua). In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human urine. These urinary adducts were enriched by a polymeric reversed phase solid-phase extraction column before the nanoLC-NSI/MS/MS analysis. The on-column detection limits (S/N≥3) of 3-EtAde and 7-EtGua were 15fg (92amol) and 10fg (56amol), respectively, while the lower quantification limits of 3-EtAde and 7-EtGua were 930 and 840 amol, respectively. Urinary concentrations of 3-EtAde and 7-EtGua in 21 smokers were 68.6±29.4 and 18.7±13.8pg/mL, respectively. In 20 nonsmokers, concentrations of 3-EtAde and 7-EtGua were 3.5±3.8 and 2.4±3.0pg/mL, respectively. The urinary concentrations of 3-EtAde and 7-EtGua were statistically significantly higher in smokers than in nonsmokers (p0.0001). Moreover, 3-EtAde and 7-EtGua concentrations are significantly correlated with the number of cigarettes smoked per day and with the smoking index. This highly specific and sensitive assay based on stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically valuable in assessing the possibility of measuring urinary ethylpurines as noninvasive biomarkers for smoking-related cancers in humans.
- Published
- 2013
20. Simultaneous quantitative analysis of N3-ethyladenine and N7-ethylguanine in human leukocyte deoxyribonucleic acid by stable isotope dilution capillary liquid chromatography–nanospray ionization tandem mass spectrometry
- Author
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Yen-Fu Liu and Hauh-Jyun Candy Chen
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,Guanine ,Adolescent ,DNA damage ,Mass spectrometry ,Tandem mass spectrometry ,Sensitivity and Specificity ,Biochemistry ,Statistics, Nonparametric ,Analytical Chemistry ,DNA Adducts ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,DNA adduct ,Leukocytes ,Humans ,Nucleotide ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Detection limit ,Chromatography ,Adenine ,Smoking ,Organic Chemistry ,Reproducibility of Results ,DNA ,General Medicine ,chemistry ,Isotope Labeling ,Female ,Quantitative analysis (chemistry) - Abstract
Cigarette smoke contains ethylating agents which damage DNA producing ethylated DNA adducts, such as N(3)-ethyladenine (3-EtAde), N(7)-ethylguanine (7-EtGua), and regioisomers of ethylthymine. Among them, 3-EtAde and 7-EtGua are present in human urine and their levels are higher in smokers than in nonsmokers. The amount of ethylated DNA adducts in tissue DNA represents the steady-state levels of DNA adducts resulting from the ethylating agent after repair in vivo. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of 3-EtAde and 7-EtGua by stable isotope dilution capillary liquid chromatography-nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS). Under the highly selective reaction monitoring (H-SRM) mode, the detection limit of 3-EtAde and 7-EtGua injected on-column was 5.0 fg (31 amol) and 10 fg (56 amol), respectively. The quantification limit for the entire assay was 50 and 100 fg of 3-EtAde and 7-EtGua, corresponding to 4.7 and 8.6 adducts in 10(9) normal nucleotides, respectively, starting with 20 μg of DNA isolated from
- Published
- 2013
21. Analysis of Ethylated Thymidine Adducts in Human Leukocyte DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography–Nanospray Ionization Tandem Mass Spectrometry
- Author
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Yi-Ching Wang, Hauh-Jyun Candy Chen, and Wen-Peng Lin
- Subjects
Detection limit ,Carbon Isotopes ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Chemistry ,Smoking ,Selected reaction monitoring ,DNA ,Urine ,Isotope dilution ,Tandem mass spectrometry ,Analytical Chemistry ,Adduct ,DNA Adducts ,chemistry.chemical_compound ,Isotope Labeling ,Leukocytes ,Humans ,Nanotechnology ,Thymidine ,Chromatography, High Pressure Liquid - Abstract
Studies showed that levels of ethylated DNA adducts in certain tissues and urine are higher in smokers than in nonsmokers. Because cigarette smoking is a major risk factor of various cancers, DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Among the ethylated DNA adducts, O(2)-ethylthymidine (O(2)-edT) and O(4)-ethylthymidine (O(4)-edT) are poorly repaired and are accumulated in the body. In addition, O(4)-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-edT adducts by isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). Under the highly selected reaction monitoring (H-SRM) mode, the detection limit of O(2)-edT, N(3)-edT, and O(4)-edT injected on-column was 5.0, 10, and 10 fg, respectively. The quantification limit for the entire assay was 50, 100, and 100 fg of O(2)-edT, N(3)-edT, and O(4)-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 10(9) normal nucleotides, respectively, starting with 50 μg of DNA (from 1.5-2.0 mL of blood). Levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 10(8) normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of O(2)-edT, N(3)-edT, and O(4)-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with pvalues of 0.0004, 0.0009, and 0.0004, respectively. Furthermore, levels of O(2)-edT show a statistically significant association (γ = 0.4789, p = 0.0327) with the smoking index in smokers. In the 40 leukocyte DNA samples, the extremely significant statistical correlations (p0.0001) are observed between levels of O(2)-edT and O(4)-edT (γ = 0.9896), between levels of O(2)-edT and N(3)-edT (γ = 0.9840), and between levels of N(3)-edT and O(4)-edT (γ = 0.9901). To our knowledge, this is the first mass spectrometry-based assay for ethylated thymidine adducts. Using this assay, the three ethylated thymidine adducts were detected and quantified for the first time. Therefore, this highly sensitive, specific, and accurate assay should be clinically feasible for simultaneous quantification of the three ethylated thymidine adducts as potential biomarkers for exposure to ethylating agents and for cancer risk assessment.
- Published
- 2012
22. Quantitative Analysis of Multiple Exocyclic DNA Adducts in Human Salivary DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography–Nanospray Ionization Tandem Mass Spectrometry
- Author
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Hauh-Jyun Candy Chen and Wen-Peng Lin
- Subjects
Saliva ,Chromatography ,DNA damage ,Indicator Dilution Techniques ,Isotope dilution ,Tandem mass spectrometry ,Salivary Glands ,Analytical Chemistry ,DNA Adducts ,chemistry.chemical_compound ,chemistry ,Deoxyadenosine ,Reference Values ,Tandem Mass Spectrometry ,Humans ,Nanotechnology ,Deoxyguanosine ,Quantitative analysis (chemistry) ,DNA ,Chromatography, Liquid - Abstract
Exocyclic DNA adducts, including 1,N(2)-propano-2'-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG) and the three lipid peroxidation-related etheno adducts 1,N(6)-etheno-2'-deoxyadenosine (edAdo), 3,N(4)-etheno-2'-deoxycytidine (edCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo), play an important role in cancer formation and they are associated with oxidative-stress-induced DNA damage. Saliva is an easily accessible and available biological fluid and a potential target of noninvasive biomarkers. In this study, a highly sensitive and specific assay based on isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) is developed for simultaneous detection and quantification of these five adducts in human salivary DNA. The levels of AdG, CdG, edAdo, edCyd, and 1,N(2)-edGuo, measured in 27 human salivary DNA samples from healthy volunteers, were determined as 104 ± 50, 7.6 ± 12, 99 ± 50, 72 ± 49, 391 ± 198 (mean ± SD) in 10(8) normal nucleotides, respectively, starting with 25 μg of DNA isolated from an average of 3 mL of saliva. Statistically significant correlations were found between levels of edAdo and edCyd (γ = 0.8007, p < 0.0001), between levels of edAdo and 1,N(2)-edGuo (γ = 0.6778, p = 0.0001), between levels of edCyd and 1,N(2)-edGuo (γ = 0.5643, p = 0.0022), between levels of AdG and 1,N(2)-edGuo (γ = 0.5756, p = 0.0017), and between levels of AdG and edAdo (γ = 0.3969, p = 0.0404). Only 5 μg of DNA sample was analyzed for simultaneous quantification of these adducts. The easy accessibility and availability of saliva and the requirement for the small amount of DNA samples make this nanoLC-NSI/MS/MS assay clinically feasible in assessing the possibility of measuring 1,N(2)-propano-2'-deoxyguanosine and etheno adducts levels in human salivary DNA as noninvasive biomarkers for DNA damage resulting from oxidative stress and for evaluating their roles in cancer formation and prevention.
- Published
- 2011
23. Analysis of Glyoxal-Induced DNA Cross-Links by Capillary Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry
- Author
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Yu-Chin Chen and Hauh-Jyun Candy Chen
- Subjects
chemistry.chemical_classification ,Detection limit ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Protein mass spectrometry ,Biomolecule ,Glyoxal ,General Medicine ,Toxicology ,Mass spectrometry ,Tandem mass spectrometry ,Adduct ,DNA Adducts ,chemistry.chemical_compound ,Cross-Linking Reagents ,chemistry ,Humans ,Spectrophotometry, Ultraviolet ,Solid phase extraction ,Chromatography, Liquid ,DNA Damage - Abstract
Glyoxal (gx) is an alpha-dicarbonyl species derived endogenously from the metabolism of carbohydrates or nitrosamines and from oxidation of lipids and nucleic acids. It is also widely distributed in foods and the environment. Glyoxal reacts with biomolecules, causing cross-links of proteins and DNA. The cross-linked products of glyoxal with 2'-deoxyribonucleosides have been characterized as dG-gx-dC, dG-gx-dG, and dG-gx-dA. We herein develop a highly specific and sensitive capillary liquid chromatography nanospray ionization tandem mass spectrometry (capLC-NSI/MS/MS) assay for the simultaneous quantification of these three DNA cross-links using a triple-quadrupole mass spectrometer. The sample pretreatment procedures included enzyme hydrolysis of DNA and adduct enrichment by a reversed phase solid phase extraction column. We compared two enzyme hydrolysis conditions, and significantly different adduct levels were observed. This assay achieved attomole sensitivity with detection limits of 12-75 amol injecting each cross-link standard on-column. After calf thymus DNA was incubated with 1.0 mM of glyoxal at 37 degrees C for 30 days, the levels of dG-gx-dC, dG-gx-dG, and dG-gx-dA in this sample were determined as 6.52, 0.80, and 2.74 in 10(5) normal nucleotides, respectively, by capLC-NSI/MS/MS analysis after hydrolysis under optimized conditions. The identity of these cross-links in glyoxal-treated DNA was confirmed by MS(2) and MS(3) scan spectra using a linear ion trap mass spectrometer. In 20 microg of human placental DNA hydrolysate, the levels of dG-gx-dC, dG-gx-dG, and dG-gx-dA were quantified as 2.49, 1.26, and 3.50 in 10(8) normal nucleotides, respectively. These DNA cross-links, if not repaired, can be mutagenic, and they represent a type of damage to the integrity of DNA structure due to exposure of glyoxal.
- Published
- 2009
24. Measurement of urinary excretion of 5-hydroxymethyluracil in human by GC/NICI/MS
- Author
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Ju-Li Huang, Hauh-Jyun Candy Chen, and Chan-Fu Wu
- Subjects
medicine.medical_specialty ,Chromatography ,DNA damage ,Urinary system ,General Medicine ,Urine ,Toxicology ,medicine.disease_cause ,Lipid peroxidation ,Excretion ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Internal medicine ,DNA adduct ,medicine ,TBARS ,Oxidative stress - Abstract
5-Hydroxymethyluracil (5-HMU) is derived from radiation in addition to endogenous oxidative DNA damage and it is one of the most abundant DNA adducts. Human 5-HMU-DNA-glycosylase has been shown to repair this lesion. Whether urinary levels of 5-HMU is a valid biomarker for oxidative DNA damage in vivo has been investigated. However, controversial results on its relation to cigarette smoking were reported. To facilitate analysis of urinary 5-HMU in epidemiological studies, a highly sensitive and specific assay based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry was developed. The limit of detection for N1,N3-bis(pentafluorobenzyl)-HMU is 10 fg (20 amol) (S/N=4) injected on column and the limit of quantification in urine was 0.7 nM of 5-HMU. Using as little as 10 microL of human urine samples, levels of urinary 5-HMU in 21 healthy volunteers were accurately quantified. No correlation was observed between urinary 5-HMU levels and cigarette smoking. However, there was a statistically significant association between urinary levels of 5-HMU and thiobarbituric acid-reactive substances (r=0.71, p=0.0003). In addition, urinary 5-HMU levels also correlated with urinary levels of 1,N6-ethenoadenine (r=0.54, p=0.01). These findings suggest that this assay should be valuable in assessing the role of urinary 5-HMU as a biomarker of oxidative DNA damage and repair.
- Published
- 2005
25. Quantification of Urinary Excretion of 1,N6-Ethenoadenine, a Potential Biomarker of Lipid Peroxidation, in Humans by Stable Isotope Dilution Liquid Chromatography−Electrospray Ionization−Tandem Mass Spectrometry: Comparison with Gas Chromatography−Mass Spectrometry
- Author
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Hauh-Jyun Candy Chen and Chia-Ming Chang
- Subjects
Detection limit ,Radioisotope Dilution Technique ,Spectrometry, Mass, Electrospray Ionization ,Chromatography ,Nitrogen Isotopes ,Adenine ,Selected reaction monitoring ,Indicator Dilution Techniques ,General Medicine ,Urine ,Isotope dilution ,Toxicology ,Mass spectrometry ,Sensitivity and Specificity ,Gas Chromatography-Mass Spectrometry ,Oxidative Stress ,chemistry.chemical_compound ,chemistry ,Humans ,Lipid Peroxidation ,Solid phase extraction ,Gas chromatography–mass spectrometry ,Derivatization ,Biomarkers ,Chromatography, High Pressure Liquid - Abstract
Etheno DNA adducts are promutagenic DNA lesions derived from exogenous as well as endogenous sources. The levels of etheno adducts in tissue DNA are elevated in cancer prone tissues, and the urinary excretion of etheno adducts is associated with oxidative stress. In this report, a new assay based on isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is developed for the quantification of 1,N(6)-ethenoadenine (epsilonAde) in human urine samples without the need for derivatization. Sample purification before analysis by MS only requires a reversed phase solid phase extraction column. Two multiple reaction monitoring transitions with two product ion fragments generated from a common parent ion were used to quantify urinary epsilonAde. The detection limit of epsilonAde using LC-ESI-MS/MS is 2 pg injected standard epsilonAde on-column, and the assay allows accurate quantification of urinary epsilonAde at concentrations higher than 10 pg/mL. The presence of epsilonAde in human urine is confirmed by the collision-induced daughter ion spectrum. Using this assay, the levels of epsilonAde in the 24 h urine samples from 18 healthy individuals are determined, and the results are in very good agreement with those obtained using isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry. The high specificity and simple sample pretreatment of this LC-ESI-MS/MS method render it a valuable tool in measuring epsilonAde in the complex mixture of human urine as a promising noninvasive biomarker for DNA damage associated with oxidative stress and for cancer chemoprevention studies.
- Published
- 2004
26. Urinary Excretion of 3,N4-Etheno-2‘-deoxycytidine in Humans as a Biomarker of Oxidative Stress: Association with Cigarette Smoking
- Author
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Chan-Fu Wu, Chia-Liang Hong, Hauh-Jyun Candy Chen, and Chia-Ming Chang
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,medicine.medical_specialty ,DNA damage ,Urinary system ,Urine ,Toxicology ,medicine.disease_cause ,Lipid peroxidation ,Cytosine ,DNA Adducts ,chemistry.chemical_compound ,Internal medicine ,medicine ,Humans ,Carcinogen ,Aged ,Creatinine ,Chemistry ,Smoking ,General Medicine ,Middle Aged ,Oxidative Stress ,Endocrinology ,Biochemistry ,Female ,Deoxycytidine ,Biomarkers ,Oxidative stress ,DNA Damage - Abstract
Smokers are known to have elevated levels of lipid peroxidation, a form of oxidative stress. Etheno DNA adduct formation can originate from endogenous lipid peroxidation or from exogenous exposure of carcinogens. Using a modified stable isotope dilution GC/negative ion chemical ionization/MS assay originally developed for urinary 3,N(4)-ethenocytosine (epsilonCyt), the nucleoside 3,N(4)-etheno-2'-deoxycytidine (epsilondCyd) was detected for the first time in human urine. The presence of epsilondCyd in human urine was confirmed by LC/electrospray ionization/tandem MS. Concentrations of epsilondCyd in the 24 h urine samples from healthy individuals not occupationally exposed to industrial chemicals were in the range between 0 and 0.80 nM. A statistically significant correlation was established between cigarette smoking and urinary excretion of epsilondCyd after being adjusted for creatinine (p = 0.004). Furthermore, the urinary total antioxidant capacity was found to correlate inversely with the epsilondCyd levels (r = -0.50, p = 0.02). The results indicate that urinary epsilondCyd may provide a valuable noninvasive biomarker for oxidative DNA damage.
- Published
- 2004
27. Detection and Quantification of 1,N6-Ethenoadenine in Human Urine by Stable Isotope Dilution Capillary Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry
- Author
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Wei-Loong Chiu and Hauh-Jyun Candy Chen
- Subjects
Adult ,Male ,Radioisotope Dilution Technique ,Spectrometry, Mass, Electrospray Ionization ,Urine ,Toxicology ,Gas Chromatography-Mass Spectrometry ,Adduct ,Excretion ,DNA Adducts ,chemistry.chemical_compound ,Humans ,Solid phase extraction ,Chemical ionization ,Creatinine ,Chromatography ,Adenine ,Smoking ,General Medicine ,Middle Aged ,Oxidative Stress ,chemistry ,DNA glycosylase ,Isotope Labeling ,Female ,Gas chromatography ,DNA Damage ,Mutagens - Abstract
1,N(6)-Ethenoadenine (epsilonAde) is a promutagenic lesion detected in tissue DNA; it has been shown that epsilonAde can be repaired by human DNA glycosylases, and it is expected to be excreted in urine. In this paper, we present for the first time detection and accurate quantification of epsilonAde in human urine samples by a highly sensitive and specific stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometric assay (GC/NICI/MS). Analysis by GC/NICI/MS includes adduct enrichment by a solid phase extraction column, followed by electrophore labeling and postderivatization cleanup. Using selective ion monitoring mode, the assay allows quantification of 0.5 pg of epsilonAde in as little as 0.1 mL of the urine sample, which is equivalent to corresponding concentration quantification limit of 31 pM. Using this assay, concentrations of epsilonAde in the 24 h urine samples of 23 healthy individuals were determined, which ranged from 0 to 124 pg/mL. After we adjusted for creatinine, a statistically significant correlation was found between epsilonAde excretion and cigarette smoking in males (p = 0.03). Thus, this stable isotope dilution GC/NICI/MS assay offers a sensitive and accurate quantification of urinary epsilonAde as a potential biomarker for oxidative damage of DNA and repair.
- Published
- 2003
28. Biological and dietary antioxidants protect against DNA nitration induced by reaction of hypochlorous acid with nitrite
- Author
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Hauh-Jyun Candy Chen, Shi-Bei Wu, and Chia-Ming Chang
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Guanine ,Hypochlorous acid ,Biophysics ,Biochemistry ,Antioxidants ,chemistry.chemical_compound ,Dihydrolipoic acid ,Nitration ,Animals ,AP site ,Nitrite ,Molecular Biology ,Nitrites ,Sulfur Compounds ,DNA ,Diet ,Hypochlorous Acid ,chemistry ,Xanthines ,Depurination ,Cattle ,Peroxynitrite ,Chromatography, Liquid ,DNA Damage - Abstract
Nitryl chloride, formed by reaction of hypochlorous acid with nitrite, might contribute to nitrative damage of biomolecules in addition to peroxynitrite. Damage of DNA by these reactive nitrogen oxide species is implicated in carcinogenesis associated with chronic infections and inflammation. Nitrated DNA adducts, such as 8-nitroguanine and 8-nitroxanthine, are not stable in DNA since they undergo spontaneous depurination, leading to apurinic site formation. In this report, we investigate the protective effect of biological and dietary antioxidants in inhibiting DNA nitration induced by nitryl chloride. The effect of inhibition was evaluated by decrease of 8-nitroxanthine and 8-nitroguanine formation. Among the 21 compounds examined, dihydrolipoic acid is the most effective in preventing DNA nitration, followed by N-acetyl-L-cysteine and folic acid. For sulfur-containing compounds, the more highly reduced compounds are stronger inhibitors of DNA nitration. The major product of N-acetyl-L-cysteine reaction with nitryl chloride is characterized as the (R)-2-acetylamino-3-sulfopropionic acid, a physiologically irreversible product, suggesting that nitryl chloride is a strong oxidizing agent.
- Published
- 2003
29. Hemoprotein-mediated reduction of nitrated DNA bases in the presence of reducing agents
- Author
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Chia-Ming Chang, Yuan Mao Chen, and Hauh-Jyun Candy Chen
- Subjects
Time Factors ,Hemeprotein ,Cytochrome ,DNA damage ,Biochemistry ,DNA Adducts ,Hemoglobins ,chemistry.chemical_compound ,Physiology (medical) ,Animals ,Humans ,Heme ,Chromatography, High Pressure Liquid ,Nitrates ,Thioctic Acid ,biology ,Chemistry ,Cytochrome c ,Cytochromes c ,DNA ,Glutathione ,Dithiothreitol ,Reducing Agents ,biology.protein ,Depurination ,Cattle ,Oxidation-Reduction ,Hemin - Abstract
DNA damages by reactive nitrogen oxide species may contribute to the multistage carcinogenesis processes associated with chronic infections and inflammation. The nitrated DNA adducts 8-nitroguanine (8NG) and 8-nitroxanthine (8NX) have been shown to derive from these reactive nitrogen oxide species, but they are not stable in DNA since they undergo spontaneous depurination. We herein report that hemin and hemoproteins, including hemoglobin and cytochrome c, mediate reduction of 8NG and 8NX to their corresponding amino analogues in the presence of reducing agents under physiologically relevant conditions. This reaction is believed to involve the reduced heme moiety produced from the reduction of oxidized hemoglobin or cytochrome c by reducing agents. The combination of hemoglobin and dihydrolipoic acid generated the reduced products in high yields. Ascorbate, quercetin, and glutathione are also capable of reducing these nitrated DNA adducts. The hemoglobin macromolecule reduces 8NG and 8NX formed in nitryl chloride-treated calf thymus DNA, as evidenced by the formation of the amino adducts using reversed-phase HPLC with photodiode array detection. Hemin is more efficient than equal molar of heme on hemoglobin in reducing 8NG-containing DNA, indicating the role of protein in impeding the reaction. Furthermore, we also show that the reduction product 8-aminoguanine is persistent on DNA. These findings suggest that reduction of nitrated DNA by the heme/antioxidant system might represent a possible in vivo pathway to modify DNA nitration.
- Published
- 2003
30. Lipoyl dehydrogenase catalyzes reduction of nitrated DNA and protein adducts using dihydrolipoic acid or ubiquinol as the cofactor
- Author
-
Chia-Ming Chang, Hauh-Jyun Candy Chen, and Yuan-Mao Chen
- Subjects
Ubiquinol ,Guanine ,Dihydrolipoamide dehydrogenase ,Thioctic Acid ,biology ,Ubiquinone ,General Medicine ,Toxicology ,Reactive Nitrogen Species ,Cofactor ,Nitroreductase ,chemistry.chemical_compound ,Biochemistry ,Dihydrolipoic acid ,chemistry ,Xanthines ,biology.protein ,Tyrosine ,Depurination ,Nitrogen Oxides ,AP site ,NAD+ kinase ,Oxidation-Reduction ,NADP ,Dihydrolipoamide Dehydrogenase - Abstract
Inflamed tissues generate reactive nitrogen oxide species (RNO(x)), such as peroxynitrite (ONOO-)and nitryl chloride (NO2Cl), which lead to formation of nitrated DNA and protein adducts, including 8-nitroguanine (8NG), 8-nitroxanthine (8NX), and 3-nitrotyrosine (3NT). Once formed, the two nitrated DNA adducts are not stable in DNA and undergo spontaneous depurination. Nitration of protein tyrosine leads to inactivation of protein functions and 3NT has been detected in various disease states. We herein report that reduction of these nitro adducts to their corresponding amino analogues can be catalyzed by lipoyl dehydrogenases (EC 1.8.1.4) from Clostridium kluyveri (ck) and from porcine heart (ph) using NAD(P)H as the cofactor. We also found that dihydrolipoic acid (DHLA) and ubiquinol can be used as effective cofactors for reduction of 8NG, 8NX, and 3NT by these lipoyl dehydrogenases. The reduction efficiency of the mammalian enzyme is higher than the bacterial isozyme. The preference of cofactors by both lipoyl dehydrogenases is DHLA>NAD(P)H>ubiquinol. In all the systems examined, the nitrated purines are reduced to a greater extent than 3NT under the same conditions. We also demonstrate that this lipoyl dehydrogenase/antioxidant system is effective in reducing nitrated purine on NO2Cl-treated double stranded calf thymus DNA, and thus decreases apurinic site formation. The nitroreductase activity for lipoyl dehydrogenase might represent a possible metabolic pathway to reverse the process of biological nitration.
- Published
- 2002
31. Role of Nitrite on Nitration of 2′-Deoxyguanosine by Nitryl Chloride
- Author
-
Yuan-Mao Chen, Hauh-Jyun Candy Chen, and Tze-Fan Wang
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,Hypochlorous acid ,chemistry ,Base (chemistry) ,Nitration ,Deoxyguanosine ,General Chemistry ,Nitrite ,Photochemistry ,Reactive nitrogen species ,Peroxynitrite ,Adduct - Abstract
Nitryl chloride and peroxynitrite are reactive nitrogen species generated by activated phagocytes against invading pathogens during infections and inflammation. In our previous report, formation of 8-nitroxanthine and 8-nitroguanine was observed in reaction of 2'-deoxyguanosine or calf thymus DNA with nitryl chloride generated by mixing hypochlorous acid (HOCI) with nitrite (NO 2 -). The present study investigates factors controlling the yields of 8-nitroxanthine and 8-nitroguanine formation in nitration of 2'-deoxyguanosine by nitryl chloride. We found that the yields of 8-nitroxanthine and 8-nitroguanine in reaction of 2'-deoxy-guanosine with nitryl chloride were highly dependent on the ratio of NO 2 - versus HOCI concentration. The yields of 8-nitroxanthine and 8-nitroguanine reached a plateau when the ratio of NO 2 - versus HOCI concentration was higher than 2. A possible mechanism was postulated to explain this observation. While 8-nitro-guanine is not stable in the presence of peroxynitrite, 8-nitroxanthine is sensitive to HOCI. The stability of these two nitrated adducts might be a factor on their final yields in this reaction. Since HOCI is produced by neutrophils at sites of inflammation where the level of NO 2 - is elevated, it is conceivable that nitryl chloride contributes to DNA base nitration in vivo, forming 8-nitroxanthine and 8-nitroguanine.
- Published
- 2002
32. 8-Nitroxanthine, an Adduct Derived from 2‘-Deoxyguanosine or DNA Reaction with Nitryl Chloride
- Author
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Kuang-Sian Wang, Tze-Fan Wang, Jentaie Shiea, Hauh-Jyun Candy Chen, and Yuan-Mao Chen
- Subjects
Magnetic Resonance Spectroscopy ,Chemistry ,Deoxyguanosine ,DNA ,General Medicine ,Hydrogen-Ion Concentration ,Toxicology ,Photochemistry ,Hypochlorous Acid ,Adduct ,DNA Adducts ,chemistry.chemical_compound ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Xanthines ,8-Nitroxanthine ,Chromatography, High Pressure Liquid ,Nitrites ,Nitryl chloride ,Reactive nitrogen species ,Peroxynitrite ,DNA Damage ,Half-Life - Abstract
Activated phagocytic cells generate reactive nitrogen species, including nitryl chloride and peroxynitrite, for host defense against invading pathogens. It has been proposed that these reactive nitrogen species may cause DNA damage and thus contribute to the multistage carcinogenesis process associated with chronic infections and inflammation. Previous studies showed that peroxynitrite reacted with guanine, 2'-deoxyguanosine, or DNA forming 8-nitroguanine. We herein report formation of 8-nitroxanthine as the major nitration product in reactions of 2'-deoxyguanosine or calf thymus DNA with nitryl chloride produced by mixing nitrite with hypochlorous acid, and 8-nitroguanine was a minor product in these reactions. 8-Nitroxanthine was characterized by its NMR and laser desorption ionization mass spectra and by deamination of 8-nitroguanine. Formation of 8-nitroxanthine was also detected by xanthine reaction with various reactive nitrogen species, including nitryl chloride, peroxynitrite, nitronium tetrafluoroborate, and heated nitric and nitrous acid. The identity of 8-nitroxanthine in nitryl chloride-treated dG and DNA was confirmed by co-injection with synthetic 8-nitroxanthine and by its reduction to 8-aminoxanthine. Levels of 8-nitroxanthine and 8-nitroguanine in these reactions were quantified by reversed-phase HPLC with photodiode array detection. Once formed, 8-nitroxanthine was spontaneously removed from DNA with a half-life of 2 h at 37 degrees C and pH 7.4. Therefore, 8-nitroxanthine might be an important DNA lesion derived from reactive nitrogen species in vivo.
- Published
- 2001
33. Detection and Quantification of 1,N6-Ethenoadenine in Human Placental DNA by Mass Spectrometry
- Author
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Lei L. Zhang, Jinsong Ni, Ming-Chung Tseng, Fung-Lung Chung, Hauh-Jyun Candy Chen, and Li-Chang Chiang
- Subjects
Adult ,Placenta ,Electrospray ionization ,Toxicology ,Mass spectrometry ,Tandem mass spectrometry ,Sensitivity and Specificity ,High-performance liquid chromatography ,Fluorescence ,Gas Chromatography-Mass Spectrometry ,DNA Adducts ,chemistry.chemical_compound ,Humans ,Chloroacetaldehyde ,Chromatography, High Pressure Liquid ,Chemical ionization ,Chromatography ,Adenine ,Reproducibility of Results ,DNA ,General Medicine ,chemistry ,Female ,Gas chromatography ,Mutagens - Abstract
Exocyclic DNA adducts have been reported to derive from various exogenous as well as endogenous sources, such as lipid peroxidation. Among them, 1,N(6)-ethenoadenine (epsilonAde) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/(32)P-postlabeling method. This study reports detection and quantification of the endogenous epsilonAde adduct in the same human placental DNA by three independent assays, namely, GC/MS, LC/MS, and HPLC/fluorescence. Using a recently reported gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method [Chen, H.-J. C., et al. (1998) Chem. Res. Toxicol. 11, 1474], the level of epsilonAde in human placental DNA from a commercial source was found to be 2.3 adducts per 10(6) Ade bases. To confirm these findings, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method was developed for epsilondAdo. With this LC/MS assay, epsilondAdo was detected at the level of 2.5 adducts per 10(6) dAdo nucleosides in the same human placental DNA. The stable isotopes of epsilonAde and epsilondAdo were added as internal standards in both GC/MS and LC/ESI/MS/MS assays, respectively, and thus provided high specificity, reproducibility, and accurate quantification. The relatively high levels of epsilonAde in this human placental DNA detected by mass spectrometry were further verified by HPLC/fluorescence analysis. The GC/MS method was validated by the HPLC/fluorescence assay using calf thymus DNA treated with chloroacetaldehyde or by the LC/MS method with 2, 3-epoxy-4-hydroxynonanal-modified calf thymus DNA. The epsilonAde level in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since epsilonAde is a potential mutagenic lesion, analysis of epsilonAde by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.
- Published
- 1999
34. DNA Adducts of 2,3-Epoxy-4-hydroxynonanal: Detection of 7-(1‘,2‘-Dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N6-Ethenoadenine by Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry
- Author
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Fung-Lung Chung, Jonathan Cox, John A. Cunningham, Lei Zhang, and Hauh-Jyun Candy Chen
- Subjects
Magnetic Resonance Spectroscopy ,Protein mass spectrometry ,Electrospray ionization ,Toxicology ,Mass spectrometry ,Gas Chromatography-Mass Spectrometry ,Adduct ,DNA Adducts ,chemistry.chemical_compound ,Animals ,Derivatization ,Chromatography, High Pressure Liquid ,Aldehydes ,Chemical ionization ,Chromatography ,Chemistry ,Adenine ,Imidazoles ,General Medicine ,Fast atom bombardment ,Rats ,Spectrometry, Fluorescence ,Liver ,Purines ,Calibration ,Epoxy Compounds ,DNA ,Mutagens - Abstract
2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids. EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EH in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1, N6-ethenoadenine (epsilonAde) and 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine (DHH-epsilonAde) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [15N5]epsilonAde and [15N5]DHH-epsilonAde to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE), (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilonAde and PFB-DHH-epsilonAde on a Si SPE column, (6) acetonide (ACT) formation of PFB-DHH-epsilonAde, and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilonAde monitoring of the (M - PFB)- ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilonAde monitoring of the (M - PFB)- ion at m/z 328 was 0.4 fmol; the detection limits for the entire assay were 6.3 fmol for epsilonAde and 36 fmol for DHH-epsilonAde. In calf thymus DNA modified with EH at 37 degreesC for 50 h, both epsilonAde and DHH-epsilonAde were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis, indicating that EH extensively reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the assay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.
- Published
- 1998
35. Synthesis of 4,6-difluoro-5-hydroxy-(α-methyl)tryptamine and 4,6-difluoro-5-hydroxy-(β-methyl)tryptamine as potential selective monoamine oxidase B inhibitors
- Author
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B. Jayachandran, Hauh-Jyun Candy Chen, Terrence Applewhite, and Kenneth L. Kirk
- Subjects
Tryptamine ,Monoamine oxidase ,Stereochemistry ,Organic Chemistry ,chemistry.chemical_element ,Ether ,Biochemistry ,Inorganic Chemistry ,chemistry.chemical_compound ,chemistry ,Fischer indole synthesis ,Nitro ,Environmental Chemistry ,Lithium ,Boron tribromide ,Monoamine oxidase B ,Physical and Theoretical Chemistry - Abstract
Condensation of 4-nitro-1-pentanal and 4-nitropentanal 3-methylbutanal with 3,5-difluoro-4-methoxyphenylhydrazine afforded 4,6-difluoro-5-methoxy-3-(2′-nitro)propylindole 4a and 4,6-difluoro-5-methoxy-3-(1′-methyl-2′-nitro)ethylindole 4b , respectively, in one step. Reduction of the nitro group with lithium aluminum hydride followed by removal of the methyl ether with boron tribromide produced the title compounds. They were inactive as MAO B inhibitors.
- Published
- 1998
36. Simultaneous Mass Spectrometric Analysis of Methylated and Ethylated Peptides in Human Hemoglobin: Correlation with Cigarette Smoking.
- Author
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Hauh-Jyun Candy Chen, Sun Wai Ip, and Fu-Di Lin
- Subjects
- *
ALKYLATING agents , *CIGARETTE smoke , *CANCER , *PROTEINS , *METHYLATION - Abstract
Alkylating agents contained in cigarettes smoke might be related to cancer development. Post-translational protein methylation and ethylation may cause alteration of protein functions. Human hemoglobin (Hb) has been a target for molecular dosimetry because of its easy accessibility. The goal of this study is to investigate the relationship between the levels of methylation and ethylation at specific sites of Hb with smoking. Because of the low extent of modification of Hb isolated from blood, the methylation and ethylation sites were identified in Hb incubated with a methylating agent (methyl methanesulfonate, MMS) and ethylating agent (ethyl methanesulfonate, EMS), respectively, by accurate mass measurements. After trypsin digestion, the modification sites were identified by nanoflow LC-nanospray ionization coupled with high-resolution mass spectrometry. The selected reaction monitoring mode was used to quantify the relative extent of methylation and ethylation in human Hb incubated with MMS and EMS, respectively. Methylation occurred at 9 sites, including ¹V, 20H, 50H, 72H of α-globin and ¹V, 26E, 66K, 77H, 93C of β-globin. Ethylation was detected at 11 sites, including ¹V, 16K, 50H, 72H, 87H of α-globin and ¹V, 17K, 66K, 77H, 92H, 93C of β-globin. The relative extents of methylation and ethylation were measured in blood samples from 13 smokers and 13 nonsmokers. No statistically significant difference was found in the methylated peptides. On the other hand, the extents of ethylation at α-terminal Val, α-His-50, α-His-87, β-terminal Val, β-His-77, and β-Cys-93 in Hb were significantly higher in smokers than in nonsmokers (p < 0.05). Furthermore, the relative extents of ethylation at these sites were statistically significantly correlated with the number of cigarettes smoked per day. Therefore, this assay, which requires as little as one drop of blood, should be helpful in measuring Hb ethylation as a potential biomarker for assessing the exposure to cigarette smoking. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
37. Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts
- Author
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Fung-Lung Chung, Hauh-Jyun Candy Chen, and Raghu G. Nath
- Subjects
Aldehydes ,Lipid Peroxides ,Cancer Research ,Mutagenesis ,Endogeny ,DNA ,General Medicine ,medicine.disease_cause ,Etheno adducts ,Rats ,Adduct ,Lipid peroxidation ,DNA Adducts ,Mice ,chemistry.chemical_compound ,Biochemistry ,chemistry ,medicine ,Animals ,Epoxy Compounds ,Humans ,Carcinogenesis ,Carcinogen - Abstract
A number of promutagenic exocyclic DNA adducts have recently been detected in both humans and rodents without carcinogen treatment. These observations raised questions about their origins and potential significance in carcinogenesis. In this commentary, we present our views pertaining to the in vivo sources of these cyclic adducts, specifically the cyclic propano and etheno adducts. The basis for our discussion comes mainly from the information generated through a span of more than a decade from several laboratories, including ours. This commentary summarizes the data from the chemical and biochemical studies that provide support for the hypothesis that lipid peroxidation is involved in the endogenous formation of these exocyclic adducts.
- Published
- 1996
38. Reactive nitrogen oxide species-induced post-translational modifications in human hemoglobin and the association with cigarette smoking
- Author
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Yu-Chin Chen and Hauh-Jyun Candy Chen
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Molecular Sequence Data ,Sulfinic acid ,Analytical Chemistry ,Nitric oxide ,chemistry.chemical_compound ,Hemoglobins ,Methionine ,Nitration ,Peroxynitrous Acid ,Humans ,Amino Acid Sequence ,Cysteine ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Smoking ,Sulfinic Acids ,Reactive Nitrogen Species ,chemistry ,Biochemistry ,Nitrosation ,Tyrosine ,Hemoglobin ,Peptides ,Oxidation-Reduction ,Protein Processing, Post-Translational ,Peroxynitrite - Abstract
Nitric oxide (NO) is essential for normal physiology, but excessive production of NO during inflammatory processes can damage the neighboring tissues. Reactive nitrogen oxide species (RNOx), including peroxynitrite (ONOO(-)), are powerful nitrating agents. Biological protein nitration is involved in several disease states, including inflammatory diseases, and it is evident by detection of 3-nitrotyrosine (3NT) in inflamed tissues. In this study, we identified peroxynitrite-induced post-translational modifications (PTMs) in human hemoglobin by accurate mass measurement as well as by the MS(2) and MS(3) spectra. Nitration on Tyr-24, Tyr-42 (α-globin), and Tyr-130 (β-globin) as well as nitrosation on Tyr-24 (α-globin) were identified. Also characterized were oxidation of all three methionine residues, α-Met-32, α-Met-76, and β-Met-55 to the sulfoxide, as well as cysteine oxidation determined as sulfinic acid on α-Cys-104 and sulfonic acid on α-Cys-104, β-Cys-93, and β-Cys-112. These modifications are detected in hemoglobin freshly isolated from human blood and the extents of modifications were semiquantified relative to the reference peptides by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the selected reaction monitoring (SRM) mode. The results showed a statistically significant positive correlation between cigarette smoking and the extents of tyrosine nitration at α-Tyr-24 and at α-Tyr-42. To our knowledge, this is the first report on identification and quantification of multiple PTMs in hemoglobin from human blood and association of a specific 3NT-containing peptide with cigarette smoking. This highly sensitive and specific assay only requires hemoglobin isolated from one drop (∼10 μL) of blood. Thus, measurement of these PTMs in hemoglobin might be feasible for assessing nitrative stress in vivo.
- Published
- 2012
39. ChemInform Abstract: Synthesis of 4,6-Difluoro-5-hydroxy-(α-methyl)tryptamine and 4,6-Difluoro-5-hydroxy-(β-methyl)tryptamine as Potential Selective Monoamine Oxidase B Inhibitors
- Author
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Hauh-Jyun Candy Chen, Kenneth L. Kirk, B. Jayachandran, and Terrence Applewhite
- Subjects
Tryptamine ,chemistry.chemical_compound ,chemistry ,Monoamine oxidase B inhibitors ,ALUMINUM HYDRIDE ,Nitro ,chemistry.chemical_element ,Lithium ,Ether ,General Medicine ,Monoamine oxidase B ,Boron tribromide ,Medicinal chemistry - Abstract
Condensation of 4-nitro-1-pentanal and 4-nitropentanal 3-methylbutanal with 3,5-difluoro-4-methoxyphenylhydrazine afforded 4,6-difluoro-5-methoxy-3-(2′-nitro)propylindole 4a and 4,6-difluoro-5-methoxy-3-(1′-methyl-2′-nitro)ethylindole 4b , respectively, in one step. Reduction of the nitro group with lithium aluminum hydride followed by removal of the methyl ether with boron tribromide produced the title compounds. They were inactive as MAO B inhibitors.
- Published
- 2010
40. Simultaneous quantification of three lipid peroxidation-derived etheno adducts in human DNA by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry
- Author
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Guan-Jih Lin, Wen-Peng Lin, and Hauh-Jyun Candy Chen
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Time Factors ,DNA damage ,Tandem mass spectrometry ,Mass spectrometry ,Hydrolysate ,Analytical Chemistry ,Lipid peroxidation ,chemistry.chemical_compound ,DNA Adducts ,Isotopes ,Enzymatic hydrolysis ,Leukocytes ,Animals ,Humans ,Nanotechnology ,Chromatography ,Isotope ,Reproducibility of Results ,Rats ,chemistry ,Calibration ,Cattle ,Female ,Lipid Peroxidation ,DNA ,Biomarkers ,Chromatography, Liquid ,DNA Damage - Abstract
Etheno DNA adducts are promutagenic DNA lesions derived from exogenous industrial chemicals, as well as endogenous sources including lipid peroxidation. Furthermore, levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues. In this study, we have developed a highly sensitive and specific stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) assay for simultaneous detection and accurate quantification of 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo), 3,N(4)-etheno-2'-deoxycytidine (epsilondCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in tissue DNA. Typically, [(13)C(1),(15)N(2)]epsilondAdo, [(15)N(3])epsilondCyd, and [(13)C(1),(15)N(2)]1,N(2)-epsilondGuo were added to calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The etheno adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by nanoLC-NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode. This nanoLC-NSI/MS/MS assay achieved attomole-level sensitivity with the detection limit of 0.73, 160, and 34 amol for the respective standard epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo injected on-column, while the quantification limit for the entire assay was 0.18, 4.0, and 3.4 fmol, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in human placental DNA were 28.2, 44.1, and 8.5 adducts in 10(8) normal nucleosides, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in 11 human leukocyte DNA samples were 16.2 +/- 5.2, 11.1 +/- 5.8, and 8.6 +/- 9.1 (mean +/- S.D.) in 10(8) normal nucleotides, respectively, starting from 30 mug of DNA or 1-1.5 mL of blood, and all the relative standard deviations were within 10%. An aliquot equivalent to 6 mug of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS/MS. Thus, this highly sensitive and specific nanoLC-NSI/MS/MS method is suitable for accurate quantification of the three lipid peroxidation-derived etheno DNA adducts as noninvasive biomarkers in clinical studies for cancer risk assessment and for evaluation of preventive agents.
- Published
- 2010
41. Simultaneous quantification of 1,N2-propano-2'-deoxyguanosine adducts derived from acrolein and crotonaldehyde in human placenta and leukocytes by isotope dilution nanoflow LC nanospray ionization tandem mass spectrometry
- Author
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Wen-Peng Lin and Hauh-Jyun Candy Chen
- Subjects
Time Factors ,Placenta ,Environmental pollution ,Clinical Chemistry Tests ,Isotope dilution ,Tandem mass spectrometry ,Analytical Chemistry ,Adduct ,Lipid peroxidation ,chemistry.chemical_compound ,Isotopes ,Pregnancy ,Tandem Mass Spectrometry ,Leukocytes ,Deoxyguanosine ,Humans ,Nanotechnology ,Crotonaldehyde ,Acrolein ,Aldehydes ,Chromatography ,Chemistry ,Reproducibility of Results ,Stereoisomerism ,DNA ,Biochemistry ,Feasibility Studies ,Female ,Biomarkers ,Chromatography, Liquid ,DNA Damage - Abstract
Humans are exposed to acrolein and crotonaldehyde due to environmental pollution and endogenous lipid peroxidation. These aldehydes react with the 2'-deoxyguanosine moiety of DNA, forming the exocyclic 1,N2-propano-2'-deoxyguanosine adducts AdG and CdG. These adducts are mutagenic lesions, and they play an important role in cancer and neurodegenerative diseases. In this study, a highly sensitive and quantitative assay was developed for simultaneous detection and quantification of AdG and CdG isomers in human placenta and leukocyte DNA by isotope dilution nanoflow LC with nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). The on-column detection limits (S/Nor = 3) of AdG and CdG were 15 and 8.9 amol, respectively. The quantification limits of AdG and CdG for the entire assay were 619 and 297 amol, respectively, corresponding to 9.8 and 4.7 adducts in 10(9) normal nucleotides, respectively, starting with 20 microg of DNA. Different enzyme hydrolysis methods were compared, and the optimal hydrolysis conditions were employed for the assay. Levels of AdG and CdG in human placental DNA (20 microg) were 108 and 26 in 10(8) normal nucleotides, respectively, with the respective relative standard deviation (RSD) of 2.6% and 3.1% (n = 3). Levels of AdG and CdG in 9 human leukocyte DNA samples were 78 +/- 23 (mean +/- SD) and 6.2 +/- 3.8 (mean +/- SD) in 10(8) normal nucleotides, respectively, starting from 30 microg of DNA. Using this assay, only 4-6 microg of DNA sample was subjected to this nanoLC-NSI/MS/MS system for analysis. Only 1-1.5 mL of blood is needed for measuring AdG and CdG levels in leukocyte DNA. Thus, it is clinically feasible using this highly sensitive assay to investigate the potential of using these adducts as noninvasive biomarkers for DNA damage resulting from acrolein and crotonaldehyde and to study their roles in cancer development and prevention.
- Published
- 2009
42. Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry
- Author
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Wei-Loong Chiu and Hauh-Jyun Candy Chen
- Subjects
Detection limit ,Radioisotope Dilution Technique ,Chromatography ,Chemistry ,General Medicine ,Urine ,Isotope dilution ,Toxicology ,Mass spectrometry ,Tandem mass spectrometry ,Sensitivity and Specificity ,Liquid chromatography–mass spectrometry ,Tandem Mass Spectrometry ,Calibration ,Humans ,Tyrosine ,Quantitative analysis (chemistry) ,Chromatography, Liquid - Abstract
Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N=3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 +/- 11.0 (mean +/- S.D.) in 10(5) tyrosine and 4.4 +/- 3.9 (mean +/- S.D.) in 10(3) tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R(2) = 0.55, p = 0.0065, n=23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.
- Published
- 2008
43. Investigation of DNA-protein cross-link formation between lysozyme and oxanine by mass spectrometry
- Author
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Hauh-Jyun Candy Chen, Wen-Peng Lin, Wei-Loong Chiu, and Siou-Siou Yang
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Guanine ,Surface Properties ,Thioester ,Tandem mass spectrometry ,Biochemistry ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Molecular Biology ,Reactive nitrogen species ,chemistry.chemical_classification ,Lysine ,Organic Chemistry ,Glutathione ,DNA ,Purine Nucleosides ,Cross-Linking Reagents ,chemistry ,Solvents ,Molecular Medicine ,Tyrosine ,Muramidase ,Ribonucleosides ,Lysozyme ,Cysteine ,Protein Binding - Abstract
Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion product originating from the guanine base through exposure to nitric oxide, nitrous acid, or N-nitrosoindoles. Oxanine was found to mediate formation of DNA-protein cross-links (DPCs) in the cell extract. We have previously characterized two DNA-protein cross-links from the reaction between Oxa and glutathione: namely, the thioester and the amide. In this study, lysozyme was used to study site-specific modification on protein by Oxa moieties in DNA. With the aid of nanoLC coupled with nanospray ionization tandem mass spectrometry, addition of Oxa was found at Lys13, Lys97, Lys116, Ser85, and Ser86 of lysozyme when it was treated with 2'-deoxyoxanosine (dOxo). Furthermore, incubation of lysozyme with Oxa-containing calf thymus DNA, produced by treating DNA with nitrous acid, led to lysozyme modification at Lys116, Ser85, and Ser86. Interestingly, none of the cysteine residues was modified by dOxo, in contrast with our previous findings that dOxo reacted with oxidized glutathione disulfide, forming the thioester. This might be due to the half-life of the dOxo-derived thioester being 2.2 days at the pH of incubation. Furthermore, the sites of modifications on lysozyme are in good agreement with the solvent accessibility of the residues. Since repair of Oxa-derived DPCs has not been extensively investigated, these results suggest that these stable DPCs might represent important forms of cellular damage caused by reactive nitrogen species involved in inflammationrelated diseases.
- Published
- 2008
44. H2O2/nitrite-induced post-translational modifications of human hemoglobin determined by mass spectrometry: redox regulation of tyrosine nitration and 3-nitrotyrosine reduction by antioxidants
- Author
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Dar-Long Cheng, Hauh-Jyun Candy Chen, Wen-Peng Lin, Mei-I Leong, and Chia-Ming Chang
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Molecular Sequence Data ,Ascorbic Acid ,Biochemistry ,Antioxidants ,chemistry.chemical_compound ,Hemoglobins ,Methionine ,Nitration ,Humans ,Trypsin ,Amino Acid Sequence ,Cysteine ,Tyrosine ,Molecular Biology ,Cysteine metabolism ,Reactive nitrogen species ,Nitrites ,Peroxidase ,Nitrates ,Nitrotyrosine ,Organic Chemistry ,Hydrogen Peroxide ,Reactive Nitrogen Species ,Vitamin B 12 ,chemistry ,Molecular Medicine ,Hemin ,Hemoglobin ,Oxidation-Reduction ,Protein Processing, Post-Translational - Abstract
Covalent modifications of proteins by endogenous reactive nitrogen oxide species lead to cytotoxic effects that are implicated in diseases associated with chronic infections and inflammation. Tyrosine nitration is a major post-translational modification of proteins by reactive nitrogen oxide species. Recent studies suggest that nitrotyrosine is not a permanent protein modification. We previously demonstrated that lipoyl dehydrogenase is capable of converting 3-nitrotyrosine into 3-aminotyrosine in the presence of certain reducing agents. In this study, we compared the abilities of various hemoproteins, hemin, and the cobalt-containing cofactor cyanocobalamin to mediate H(2)O(2)/nitrite-dependent tyrosine nitration and found that these hemoproteins and metal-containing cofactors also catalyzed the reduction of 3-nitrotyrosine to various extents in the presence of thiol reducing agents or ascorbate. The H(2)O(2)/nitrite-induced post-translational modifications of human hemoglobin identified by nanoLC/nanospray ionization tandem mass spectrometric analysis of the tryptic digest include nitration of tyrosine and tryptophan, as well as oxidation of methionine and cysteine residues. Nitration of human hemoglobin by H(2)O(2)/nitrite was detected on Tyr24 and Tyr42 (alpha-chain) and on Tyr130 and Trp15 (beta-chain) in the alphabeta-dimer. Oxidation of methionine and cysteine residues was also observed. Furthermore, hemoglobin also catalyzed nitro reduction of 3-nitrotyrosine to form 3-aminotyrosine, at Tyr24 in the alpha-chain peptide of human Hb in the presence of ascorbate. The enhanced peroxidase activity of nitrated hemoglobin can be reversed by the antioxidant ascorbate. These results suggest a possible in vivo pathway for hemoglobin contributing to denitration of nitrated proteins through redox regulation.
- Published
- 2007
45. Characterization of DNA--protein cross-links induced by oxanine: cellular damage derived from nitric oxide and nitrous acid
- Author
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Li-Ching Shen, Hauh-Jyun Candy Chen, Chia-Jong Hsieh, and Chia-Ming Chang
- Subjects
Guanine ,Deoxyribonucleosides ,Nitrous Acid ,Tripeptide ,Thioester ,Nitric Oxide ,Biochemistry ,Nitric oxide ,chemistry.chemical_compound ,Tandem Mass Spectrometry ,Nuclear Magnetic Resonance, Biomolecular ,Reactive nitrogen species ,Chromatography, High Pressure Liquid ,Gel electrophoresis ,chemistry.chemical_classification ,Glutathione Disulfide ,Chemistry ,Proteins ,Glutathione ,DNA ,Purine Nucleosides ,Reactive Nitrogen Species ,Cross-Linking Reagents - Abstract
Reactive nitrogen species are implicated in inflammatory diseases and cancers. Oxanine (Oxa) is a DNA lesion derived from the guanine base with nitric oxide, nitrous acid, or N-nitrosoindoles. It was shown by gel electrophoresis that oxanine mediated the formation of DNA-protein cross-links (DPCs) with DNA-binding proteins and in the cell extract. Although 2'-deoxyoxanosine was shown to react with amines including the N-terminal amino group of glycine, the structures of DNA-protein cross-links induced by oxanine have not been characterized. In this study, we find that the thiol group of the amino acid side chain is reactive toward oxanine, forming a thioester. Two reaction products of oxanine, namely, the thioester and the amide adducts, with the endogenous tripeptide glutathione (GSH) as a model protein were characterized on the basis of their UV, NMR (1H- and 13C-), and mass spectra. Interestingly, the disulfide GSSG also reacts with oxanine, forming the thioester adduct. The thioester and the amide adducts are generated when GSH and GSSG react with oxanine-containing calf thymus DNA, and they might be possible forms of cellular DPCs. Because the repair mechanism of DPCs is not extensively investigated, the characterization of oxanine-derived DPC structures should shed light on their detection in vivo and on their biological consequences.
- Published
- 2007
46. Association between cigarette smoking and urinary excretion of 1,N2-ethenoguanine measured by isotope dilution liquid chromatography-electrospray ionization/tandem mass spectrometry
- Author
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Wei-Loong Chiu and Hauh-Jyun Candy Chen
- Subjects
Adult ,Male ,Spectrometry, Mass, Electrospray Ionization ,Guanine ,Indicator Dilution Techniques ,Urine ,Isotope dilution ,Toxicology ,Tandem mass spectrometry ,chemistry.chemical_compound ,DNA Adducts ,DNA adduct ,Humans ,Detection limit ,Creatinine ,Chromatography ,Selected reaction monitoring ,Smoking ,Reproducibility of Results ,General Medicine ,Middle Aged ,Triple quadrupole mass spectrometer ,chemistry ,Female ,Biomarkers ,Chromatography, Liquid - Abstract
Levels of the promutagenic 1,N2-ethenoguanine (1,N2-epsilonGua), an etheno DNA adduct derived mainly from lipid peroxidation, in experimental animals are associated with risk of cancer formation. Since 1,N2-epsilonGua can be repaired by human glycosylases, it is possible to use it as a biomarker for cancer risk assessment in humans. In the present study, a highly sensitive and specific stable isotope dilution liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) was developed for accurate quantification of 1,N2-epsilonGua in human urine. The sample pretreatment involved a consecutive strong cation exchange solid-phase extraction (SPE) and reversed phase SPE chromatography. The pretreated sample was analyzed by LC-ESI/MS/MS under multiple reaction monitoring mode (MRM) using a triple quadrupole mass spectrometer. The detection limit of 1,N2-epsilonGua using this LC-ESI/MS/MS assay was 1.0 pg (5.8 fmol) injected on-column. Levels of urine samples collected from healthy volunteers were found to range from 0 to 199 pg/mL, and levels as low as 5.0 pg/mL (29 pM) could be accurately quantified. After adjusting for creatinine levels and body weight, an statistically significant association was observed between urinary levels of 1,N2-epsilonGua and cigarette smoking (p = 0.0006). This highly specific and sensitive assay should be valuable in measuring urinary 1,N2-epsilonGua as a potential noninvasive biomarker for oxidative DNA damage.
- Published
- 2006
47. Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry.
- Author
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Hauh-Jyun Candy Chen, Chih-Huang Fan, and Ya-Fen Yang
- Subjects
- *
LIQUID chromatography , *REACTIVE nitrogen species , *HEMOGLOBINS , *TANDEM mass spectrometry , *SMOKING , *DIABETES - Abstract
Dried blood spot (DBS) is an emerging microsampling technique for the bioanalysis of small molecules, including fatty acids, metabolites, drugs, and toxicants. DBS offers many advantages as a sample format including easy sample collection and cheap sample shipment. Hemoglobin adducts have been recognized as a suitable biomarker for monitoring chemical exposure. We previously reported that certain modified peptides in hemoglobin derived from reactive chlorine, nitrogen, and oxygen species are associated with factors including smoking, diabetes mellitus, and aging. However, the stability of these oxidation-induced modifications of hemoglobin remains unknown and whether they can be formed artifactually during storage of DBS. To answer these questions, globin extracted from the DBS cards was analyzed, and the stability of the modifications was evaluated. After storage of the DBS cards at 4 °C or room temperature up to 7 weeks, we isolated globin from a quarter of the spot every week. The extents of 11 sites and types of post-translational modifications (PTMs), including nitration and nitrosylation of tyrosine and oxidation of cysteine and methionine residues, in human hemoglobin were measured in the trypsin digest by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) using selected reaction monitoring. The extents of all these PTMs are stable within 14 days when stored on DBS at room temperature and at 4 °C, while those from direct extraction of fresh blood are stable for at least 8 weeks when stored as an aqueous solution at -20 °C. Extraction of globin from a DBS card is of particular importance for hemolytic blood samples. To our knowledge, this is the first report on the stability of oxidative modifications of hemoglobin on DBSs, which are stable for 14 days under ambient conditions (room temperature, in air). Therefore, it is feasible and convenient to analyze these hemoglobin modifications from DBSs in studies involving large populations. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
48. Measurement of urinary excretion of 5-hydroxymethyluracil in human by GC/NICI/MS: correlation with cigarette smoking, urinary TBARS and etheno DNA adduct
- Author
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Hauh-Jyun Candy, Chen, Chan-Fu, Wu, and Ju-Li, Huang
- Subjects
Adult ,Male ,Adenine ,Smoking ,Middle Aged ,Thiobarbituric Acid Reactive Substances ,Gas Chromatography-Mass Spectrometry ,Fluorobenzenes ,Pentoxyl ,DNA Adducts ,Humans ,Female ,Lipid Peroxidation ,Biomarkers ,Aged ,DNA Damage - Abstract
5-Hydroxymethyluracil (5-HMU) is derived from radiation in addition to endogenous oxidative DNA damage and it is one of the most abundant DNA adducts. Human 5-HMU-DNA-glycosylase has been shown to repair this lesion. Whether urinary levels of 5-HMU is a valid biomarker for oxidative DNA damage in vivo has been investigated. However, controversial results on its relation to cigarette smoking were reported. To facilitate analysis of urinary 5-HMU in epidemiological studies, a highly sensitive and specific assay based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry was developed. The limit of detection for N1,N3-bis(pentafluorobenzyl)-HMU is 10 fg (20 amol) (S/N=4) injected on column and the limit of quantification in urine was 0.7 nM of 5-HMU. Using as little as 10 microL of human urine samples, levels of urinary 5-HMU in 21 healthy volunteers were accurately quantified. No correlation was observed between urinary 5-HMU levels and cigarette smoking. However, there was a statistically significant association between urinary levels of 5-HMU and thiobarbituric acid-reactive substances (r=0.71, p=0.0003). In addition, urinary 5-HMU levels also correlated with urinary levels of 1,N6-ethenoadenine (r=0.54, p=0.01). These findings suggest that this assay should be valuable in assessing the role of urinary 5-HMU as a biomarker of oxidative DNA damage and repair.
- Published
- 2004
49. Effect of cigarette smoking on urinary 3,N4-ethenocytosine levels measured by gas chromatography/mass spectrometry
- Author
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Wei-Loong Chiu, Hauh-Jyun Candy Chen, Chia-Liang Hong, and Chan-Fu Wu
- Subjects
Adult ,medicine.medical_specialty ,Creatinine ,Chromatography ,Smoking ,Urine ,Isotope dilution ,Middle Aged ,Toxicology ,medicine.disease_cause ,Mass spectrometry ,Gas Chromatography-Mass Spectrometry ,Lipid peroxidation ,chemistry.chemical_compound ,Cytosine ,DNA Adducts ,Endocrinology ,chemistry ,Internal medicine ,medicine ,Humans ,Gas chromatography–mass spectrometry ,Oxidative stress ,Carcinogen ,Aged - Abstract
Etheno DNA adducts are DNA damages derived from exogenous carcinogens as well as endogenous lipid peroxidation and oxidative stress. Elevated levels of etheno DNA adducts were found in cancer-prone tissues and blood samples, suggesting that these promutagenic lesions correlate with risk of cancers. We previously reported the detection of 3,N4-ethenocytosine (epsilon Cyt) in the urine samples of two smokers using the isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay (Chen et al., 2001, Chem. Res. Toxicol. 14, 1612-1619). Since smokers are found to have elevated levels of lipid peroxidation and oxidative stress, we examined the association between urinary epsilon Cyt levels with cigarette smoking. Among the 23 samples analyzed, the average concentration of urinary epsilon Cyt in smokers was significantly higher than that of nonsmokers, 2.65 +/- 4.0 versus 0.61 +/- 0.90 ng/kg/g creatinine (p= 0.03). Albeit the number of subjects is limited, the results indicate that the measurement of epsilon Cyt in human urine may provide a useful noninvasive biomarker for oxidative DNA damage and cancer chemoprevention studies.
- Published
- 2003
50. Detection and quantification of 5-chlorocytosine in DNA by stable isotope dilution and gas chromatography/negative ion chemical ionization/mass spectrometry
- Author
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Hauh-Jyun Candy Chen, Chia-Liang Hong, and Shin-Wei Row
- Subjects
Detection limit ,Chemical ionization ,Carbon Isotopes ,Chromatography ,Nitrogen Isotopes ,Chemistry ,Indicator Dilution Techniques ,General Medicine ,Isotope dilution ,Toxicology ,Mass spectrometry ,High-performance liquid chromatography ,Gas Chromatography-Mass Spectrometry ,chemistry.chemical_compound ,Cytosine ,DNA Adducts ,Animals ,Humans ,Selected ion monitoring ,Cattle ,Gas chromatography ,DNA - Abstract
Hypochlorous acid (HOCl) is generated from activated phagocytes during infections and inflammation. One of the major products of HOCl reaction with DNA was 5-chlorocytosine (5Cl-Cyt). In this report, a gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) assay with stable isotope dilution was developed for detection and quantification of 5Cl-Cyt in DNA. During hydrolysis of DNA, 5Cl-Cyt undergoes spontaneous deamination quantitatively forming 5-chlorouracil (5Cl-Ura). The stable isotope of 5Cl-Ura with six mass units higher than the normal 5Cl-Ura was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selected ion monitoring at [M - 181](-) fragments of bispentafluorobenzylated 5Cl-Ura and its isotope analogue. The limit of detection was 20 amol (S/N = 8) of bispentafluorobenzylated 5Cl-Ura injected on column with selective ion monitoring mode and the limit of quantification for the entire assay was 14 fmol of 5Cl-Cyt. Analysis of hypochlorous acid-treated calf thymus DNA by both GC/NICI/MS and HPLC/UV detection provided similar adduct levels and thus verified this new GC/NICI/MS assay. Using this highly specific and ultrasensitive GC/NICI/MS method, the levels of 5Cl-Cyt in untreated calf thymus DNA and human placental DNA were determined as 0.6 and 6.6 adducts per 10(7) normal cytosine, respectively. Peroxynitrite also contributed to 5Cl-Cyt formation in DNA. Level of 5Cl-Cyt in DNA treated with peroxynitrite in the presence of chloride was higher than that without addition of chloride. Thus, quantification of 5Cl-Cyt in DNA by this isotope dilution GC/NICI/MS assay may facilitate research on the role of DNA chlorination in carcinogenesis and in cancer development.
- Published
- 2002
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