Nanoflow Liquid Chromatography-nanospray Ionization Research Articles

Correlation between Glyoxal-Induced DNA Cross-Links and Hemoglobin Modifications in Human Blood Measured by Mass Spectrometry.

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.

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.

Analysis of Chlorination, Nitration, and Nitrosylation of Tyrosine and Oxidation of Methionine and Cysteine in Hemoglobin from Type 2 Diabetes Mellitus Patients by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

The post-translational modification (PTM) of proteins by endogenous reactive chlorine, nitrogen, and oxygen species is implicated in certain pathological conditions, including diabetes mellitus. Evidence showed that the extents of modifications on a number of proteins are elevated in diabetic patients. Measuring modification on hemoglobin has been used to monitor the extent of exposure. This study develops an assay for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin and to examine whether the level of any of these modifications is higher in poorly controlled type 2 diabetic mellitus patients. This mass spectrometry-based assay used the bottom-up proteomic strategy. Due to the low amount of endogenous modification, we first characterized the sites of chlorination at tyrosine in hypochlorous acid-treated hemoglobin by an accurate mass spectrometer. The extents of chlorination, nitration, and oxidation of a total of 12 sites and types of modifications in hemoglobin were measured by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry under the selected reaction monitoring mode. Relative quantification of these PTMs in hemoglobin extracted from blood samples shows that the extents of chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in diabetic patients (n = 19) than in nondiabetic individuals (n = 18). After excluding the factor of smoking, chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in the nonsmoking diabetic patients (n = 12) than in normal nonsmoking subjects (n = 11). Multiple regression analysis performed on the combined effect of age, body-mass index (BMI), and HbA1c showed that the diabetes factor HbA1c contributes significantly to the extent of chlorination at α-Tyr-24 in nonsmokers. In addition, age contributes to oxidation at α-Met-32 significantly in all subjects and in nonsmokers. These results suggest the potential of using chlorination at α-Tyr-24-containing peptide to evaluate protein damage in nonsmoking type 2 diabetes mellitus.

Multistage Mass Spectrometric Analysis of Human Hemoglobin Glutathionylation: Correlation with Cigarette Smoking

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 and <0.0001, 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.

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.

Reactive Nitrogen Oxide Species-Induced Post-Translational Modifications in Human Hemoglobin and the Association with Cigarette Smoking

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.

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.