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 MS² and MS³ 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

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,<i>N</i>²-propano-2′-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG) and the three lipid peroxidation-related etheno adducts 1,<i>N</i>⁶-etheno-2′-deoxyadenosine (εdAdo), 3,<i>N</i>⁴-etheno-2′-deoxycytidine (εdCyt), and 1,<i>N</i>²-etheno-2′-deoxyguanosine (1,<i>N</i>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,<i>N</i>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 10⁸ 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, <i>p</i> < 0.0001), between levels of εdAdo and 1,<i>N</i>²-εdGuo (γ = 0.6778, <i>p</i> = 0.0001), between levels of εdCyd and 1,<i>N</i>²-εdGuo (γ = 0.5643, <i>p</i> = 0.0022), between levels of AdG and 1,<i>N</i>²-εdGuo (γ = 0.5756, <i>p</i> = 0.0017), and between levels of AdG and εdAdo (γ = 0.3969, <i>p</i> = 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,<i>N</i>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 Mass Spectrometric Analysis of Methylated and Ethylated Peptides in Human Hemoglobin: Correlation with Cigarette Smoking

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 (<i>p</i> < 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

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 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, <i>O</i>²-ethylthymidine (<i>O</i>²-edT) and <i>O</i>⁴-ethylthymidine (<i>O</i>⁴-edT) are poorly repaired and are accumulated in the body. In addition, <i>O</i>⁴-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of <i>O</i>²-edT, <i>N</i>³-edT (<i>N</i>³-ethylthymidine), and <i>O</i>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 <i>O</i>²-edT, <i>N</i>³-edT, and <i>O</i>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 <i>O</i>²-edT, <i>N</i>³-edT, and <i>O</i>⁴-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 10⁹ normal nucleotides, respectively, starting with 50 μg of DNA (from 1.5–2.0 mL of blood). Levels of <i>O</i>²-edT, <i>N</i>³-edT, and <i>O</i>⁴-edT in 20 smokers’ leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 10⁸ normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of <i>O</i>²-edT, <i>N</i>³-edT, and <i>O</i>⁴-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with <i>p</i> values of 0.0004, 0.0009, and 0.0004, respectively. Furthermore, levels of <i>O</i>²-edT show a statistically significant association (γ = 0.4789, <i>p</i> = 0.0327) with the smoking index in smokers. In the 40 leukocyte DNA samples, the extremely significant statistical correlations (<i>p</i> < 0.0001) are observed between levels of <i>O</i>²-edT and <i>O</i>⁴-edT (γ = 0.9896), between levels of <i>O</i>²-edT and <i>N</i>³-edT (γ = 0.9840), and between levels of <i>N</i>³-edT and <i>O</i>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

Mass Spectrometric Analysis of Glyoxal and Methylglyoxal-Induced Modifications in Human Hemoglobin from Poorly Controlled Type 2 Diabetes Mellitus Patients

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 MS² and MS³ 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 (<i>n</i> = 20) and in nondiabetic control subjects (<i>n</i> = 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 (<i>p</i> value <0.05). This report identified and quantified glyoxal- and methylglyoxal-modified hemoglobin peptides in humans and revealed the association of the extent of modifications at specific sites with T2DM. Only one drop (10 μL) of fresh blood is needed for this assay, and only an equivalent of 1 μg of hemoglobin was analyzed by the nanoLC-NSI/MS/MS-SRM system. These results suggest the potential use of these specific post-translational modifications in hemoglobin as feasible biomarker candidates to assess protein damage induced by glyoxal and methylglyoxal

Mass Spectrometric Analysis of Glyoxal and Methylglyoxal-Induced Modifications in Human Hemoglobin from Poorly Controlled Type 2 Diabetes Mellitus Patients

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 MS² and MS³ 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 (<i>n</i> = 20) and in nondiabetic control subjects (<i>n</i> = 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 (<i>p</i> value <0.05). This report identified and quantified glyoxal- and methylglyoxal-modified hemoglobin peptides in humans and revealed the association of the extent of modifications at specific sites with T2DM. Only one drop (10 μL) of fresh blood is needed for this assay, and only an equivalent of 1 μg of hemoglobin was analyzed by the nanoLC-NSI/MS/MS-SRM system. These results suggest the potential use of these specific post-translational modifications in hemoglobin as feasible biomarker candidates to assess protein damage induced by glyoxal and methylglyoxal