Chromatography Nanospray Ionization Tandem Mass Research Articles

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

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]dG-gx-dC and [15N5]dG-gx-dA as internal standards, enzyme hydrolysis to release the cross-links as nucleosides, enrichment by a reversed-phase solid-phase extraction column, and nanoLC-NSI/MS/MS analysis. The detection limit is 0.19 amol for dG-gx-dC and 0.89 amol for dG-gx-dA, which is 400 and 80 times more sensitive, respectively, than capillary LC-NSI/MS/MS assay of these adducts. The lower limit of quantification was 94 and 90 amol for dG-gx-dC and dG-gx-dA, respectively, which is equivalent to 0.056 and 0.065 adducts in 108 normal nucleotides in 50 μg of DNA. In type 2 diabetes mellitus (T2DM) patients (n = 38), the levels of dG-gx-dC and dG-gx-dA in leukocyte DNA were 1.94 ± 1.20 and 2.10 ± 1.77 in 108 normal nucleotides, respectively, which were significantly higher than those in nondiabetics (n = 39: 0.83 ± 0.92 and 1.05 ± 0.99 in 108 normal nucleotides, respectively). Excluding the factor of smoking, an exogenous source of glyoxal, levels of these two cross-linked adducts were found to be significantly higher in nonsmoking T2DM patients than in nonsmoking control subjects. Furthermore, the levels of dG-gx-dC and dG-gx-dA correlated with HbA1c with statistical significance. To our best knowledge, this is the first report of the identification and quantification of glyoxal-derived cross-linked DNA adducts in human leukocyte DNA and their association with T2DM. This SID nanoLC-NSI/MS/MS assay is highly sensitive and specific and it requires only 50 μg of leukocyte DNA isolated from 2-3 mL of blood to accurately quantify these two cross-linked adducts simultaneously. Our assay thus provides a useful biomarker for the evaluation of glyoxal-derived DNA damage.

Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry.

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.

Noninvasive measurement of smoking-associated N3-ethyladenine and N7-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry

Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N3-ethyladenine (3-EtAde) and N7-ethylguanine (7-EtGua). These two ethylpurines can be depurinated spontaneously and be repaired by enzymes and they have been detected in human urine. 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 salivary DNA. These ethylpurines were released from DNA by neutral thermal hydrolysis and then enriched by a solid-phase extraction column before nanoLC–NSI/MS/MS analysis. The detection limits (S/N≥3) of 3-EtA and 7-EtG were 15fg (92amol) and 10fg (56amol), respectively, injected on-column. The lower quantification limits of 3-EtAde and 7-EtGua were both 100fg, i.e. 620 and 560amol, respectively, corresponding to 9.4 and 8.6 adducts in 109 normal nucleotides, respectively, starting with as little as 20μg of DNA isolated from an average of 3mL of saliva. The mean (±SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6±7.0 and 9.7±5.3 in 108 normal nucleotides, respectively, while those of 7-EtGua were 14.1±8.2 and 3.8±2.8 in 108 normal nucleotides in smokers and nonsmokers, respectively. Levels of 7-EtGua, but not 3-EtAde, were statistically significantly higher in smokers than in nonsmokers (p<0.0001). Furthermore, salivary 7-EtGua levels are significantly correlated with the number of cigarettes smoked per day as well as with the smoking index. This highly specific and sensitive stable isotope dilution nanoLC–NSI/MS/MS assay might be feasible in measuring 7-EtGua in human salivary DNA as a noninvasive biomarker for DNA damage induced by cigarette smoking.

Simultaneous quantification of ethylpurine adducts in human urine by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry

Ethylating agents contained in cigarette smoke can damage DNA producing ethylated DNA adducts, including N3-ethyladenine (3-EtAde) and N7-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 (p<0.0001). 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.

Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry

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, O2-ethylthymidine (O2-edT) and the promutagenic O4-ethylthymidine (O4-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), O2-edT, N3-edT (N3-ethylthymidine), and O4-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 50μg of DNA isolated from about 3.5mL of saliva, levels of O2-edT, N3-edT, and O4-edT in 20 smokers’ salivary DNA samples were 5.3±6.2, 4.5±5.7, 4.2±8.0 in 108 normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p<0.0001) were observed between levels of O2-edT and N3-edT (γ=0.7388), between levels of O2-edT and O4-edT (γ=0.8839), and between levels of N3-edT, and O4-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.

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

Cigarette smoke contains ethylating agents which damage DNA producing ethylated DNA adducts, such as N3-ethyladenine (3-EtAde), N7-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 5.0fg (31amol) and 10fg (56amol), respectively. The quantification limit for the entire assay was 50 and 100fg of 3-EtAde and 7-EtGua, corresponding to 4.7 and 8.6 adducts in 109 normal nucleotides, respectively, starting with 20μg of DNA isolated from <1mL of blood and injecting an equivalent of 4μg of DNA on-column. The mean (±SD) levels of 3-EtAde and 7-EtGua in leukocyte DNA from 20 smokers were 16.0±7.8 and 9.7±8.3 in 108 normal nucleotides, respectively, which were statistically significantly higher than those of 5.4±2.6 3-EtAde and 0.3±0.8 7-EtGua in 108 normal nucleotides from 20 nonsmokers (p<0.0001). The levels of 3-EtAde and 7-EtGua in these 40 leukocyte DNA samples are positively correlated (γ=0.6970, p<0.0001). Furthermore, there are statistically significant associations between the number of cigarettes smoked per day, as well as the smoking index, and the levels of 3-EtAde and 7-EtGua. Levels of 3-EtAde and 7-EtGua are compared to those of ethylthymidine adducts. To our knowledge, this is the first assay for simultaneous quantification of 3-EtAde and 7-EtGua in the same DNA sample and is the first report of 3-EtAde in human DNA. This highly sensitive and specific stable isotope dilution capLC–NSI/MS/MS assay should be useful in measuring 3-EtAde and 7-EtGua in human leukocyte DNA as potential biomarkers for smoking-related cancers.

Protein expression in pectoral skeletal muscle of chickens as influenced by dietary methionine

Effects of dietary methionine (Met) on pectoralis muscle development and the effect that Met as a nutritional substrate has on protein expression of skeletal muscle cells of pectoralis muscle of chickens were evaluated in this study. Broiler chickens received a common pretest diet up to 21 d of age and were subsequently fed either a low (LM) or high Met (HM) diet (0.41 vs. 0.51% of diet) from 21 to 42 d of age. Dietary deficiency was shown in vivo judging by the depression in breast meat weight and yield when broilers were fed the LM diet. Global protein expression was analyzed by quantitative high-performance liquid chromatography nanospray ionization tandem mass spectrometry. Up- and downregulated proteins were analyzed via Ingenuity Pathways Analysis to identify the metabolic pathways affected. Four canonical pathways related to muscle development were identified as being differentially regulated between LM- and HM-fed chickens. These pathways included the citrate cycle and calcium, actin cytoskeleton, and clathrin-mediated endocytosis signaling. The HM diet may have allowed for increased muscle growth by an increased availability of nutrients to muscle cells. Although the Met supplementation was associated with enhanced breast muscle growth, contraction fiber concentrations in muscles decreased and were associated with a lower calcium transportation rate and sensitivity and with a lower energy supply. It is further suggested that increased muscle protein deposition, that was induced by Met supplementation, may have been largely due to sarcoplasmic rather myofibrillar hypertrophy.

Analysis of Ethylated Thymidine Adducts in Human Leukocyte DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography–Nanospray Ionization Tandem Mass Spectrometry

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 (p < 0.0001) 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.

Quantitative Analysis of Multiple Exocyclic DNA Adducts in Human Salivary DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography–Nanospray Ionization Tandem Mass Spectrometry

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 (εdAdo), 3,N(4)-etheno-2'-deoxycytidine (εdCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdGuo), 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, εdAdo, εdCyd, and 1,N(2)-εdGuo, 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 εdAdo and εdCyd (γ = 0.8007, p < 0.0001), between levels of εdAdo and 1,N(2)-εdGuo (γ = 0.6778, p = 0.0001), between levels of εdCyd and 1,N(2)-εdGuo (γ = 0.5643, p = 0.0022), between levels of AdG and 1,N(2)-εdGuo (γ = 0.5756, p = 0.0017), and between levels of AdG and εdAdo (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.

Simultaneous Quantification of Three Lipid Peroxidation-Derived Etheno Adducts in Human DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

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.

Analysis of Glyoxal-Induced DNA Cross-Links by Capillary Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

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.