Article pubs.acs.org/crt
Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry Hauh-Jyun Candy Chen,* Chih-Huang Fan, and Ya-Fen Yang Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan S Supporting Information *
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.
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INTRODUCTION Blood sampling by the dried blood spot (DBS) technique, i.e., pricking the finger, toe, or heel with a lancet and blotting blood on filter paper, was first used in newborns for phenylketonuria, an inherited disease.1 This technique is now commonly applied to newborn screening and it has other clinical applications.2,3 Advantages in using dried blood spot (DBS) over whole blood samples include small sample volume as well as easy sample collection, storage, and shipment. In addition, DBS is a minimally invasive technique and is an ideal self-sampling method for mass screening of clinical research comprising large populations.4 Drugs and metabolites preserved within a dried blood spot can be stable for years at ambient conditions5 and can be eluted in solvents for later analysis. Furthermore, infectious pathogens, such as blood borne HIV virus, are deactivated upon drying in the filter paper, and there are thus fewer biohazards associated with DBS than with whole blood.3 The DBS technology has expanded and is considered a “green” technology due to the reduced amount of specimen, labware, and energy needed compared to those of handling whole blood.6 Combination of DBS with liquid chromatography−mass © XXXX American Chemical Society
spectrometry (LC−MS) is valuable in analyzing small molecules, such as fatty acids, nutrients, and metabolites, therapeutic drugs, carcinogens, and toxicants.7,8 Reactive species from exogenous and endogenous sources can damage DNA and proteins causing detrimental biological effects. Currently, the levels of DNA and protein adducts are used for assessing the biologically effective dose of these reactive species in the tissues. However, it is difficult to obtain DNA and protein from the target tissues of humans and a surrogate such as peripheral blood is thus used. In one milliliter of blood, there are only several micrograms of DNA, but there are ca. 150 mg of hemoglobin (Hb) and 30 mg of serum albumin (SA).9 Although DNA damage is directly associated with carcinogenesis, levels of DNA adducts are much smaller than those of protein adducts. Linear dose−response curves were found for both DNA and Hb adducts in animals and in humans exposed to carcinogens.10−12 Protein adducts are not repaired and are much more abundant Received: September 21, 2016 Published: November 8, 2016 A
DOI: 10.1021/acs.chemrestox.6b00334 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology than DNA adducts in blood.9 Thus, Hb adducts can be a surrogate for DNA adducts in molecular dosimetry.13,14 Traditionally, levels of Hb adducts have been analyzed by the modified Edman degradation method, followed by gas chromatography−mass spectrometry (GC−MS) analysis.15 Recently, the Edman degradation products of the N-terminal valine residues were analyzed by LC−MS.16,17 Using Hb adduct derived from benzene oxide, Funk and co-workers demonstrated that protein adducts can be measured in DBSs, and the adduct levels in hemoglobin obtained from DBS or conventional red blood cells are correlated.18 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated during aerobic metabolism, and cells contain antioxidant defense systems to balance their production. Overproduction of these reactive species, leading to oxidative stress, is implicated in many diseases, including cancer. Post-translational modification (PTM) of proteins plays an imperative role in protein activity, function, and regulation.19,20 The development of mass spectrometry-based technology has empowered advancement of biomedical research in redox proteomics.21−23 Quantitation of trace amounts of PTM in proteins is a demanding task. The concept of “native reference peptide (NRP)” was originally described by Ruse et al. for phosphopeptides.24 The unmodified peptide present in the protein digest is used as the reference peptide for relative quantification of PTM. The advantages of this method include correcting for variations in protein amounts and peptide recovery in the digestion procedure and the broad applicability to a variety of PTMs. In addition, there is no need to use the costly stable isotope labeled-peptides. Using this approach, we reported that certain peptides in hemoglobin are modified by oxidative species leading to nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of cysteine and methionine, and glutathionylation of cysteine residues.25−28 The levels of these PTMs are correlated with factors such as smoking, diabetes mellitus, aging, or body-mass index.25,27,28 It is not known whether nitration and oxidation in hemoglobin can form artifactually during the storage of DBS in air. Using a proteomic approach with liquid chromatography− tandem mass spectrometry (LC−MS/MS), the derivatization step can be omitted, and modification on multiple sites can be analyzed simultaneously. In this study, we analyzed globin extracted from the DBS cards and evaluated the stability of these oxidative stress-induced hemoglobin modifications by nanoflow liquid chromatography−nanospray ionization tandem mass spectrometry (nanoLC−NSI/MS/MS).
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Isolation of Human Globin from Dried Blood Spots. The following procedures were modified from the procedures reported by Funk et al.18 The dried blood spot card was cut with cleaned scissors to 1/2, 1/4, 1/8, or 1/16 to determine the extraction efficiency of hemoglobin. One milliliter of deionized water was added to a portion of the DBS card in a vial and allowed it to shake at 160 rpm for 90 min. After the card was removed with tweezers, the solution was filtered by a 0.22 μm Nylon syringe filter to remove cell debris and concentrated by a centrifugal vacuum concentrator to 0.2 mL, followed by addition of 1 mL of 43% ethanol (v/v) with vigorous shaking. The solution was left at −20 °C for 1 h, transferred to a 1.5 mL Eppendorf tube, followed by centrifugation at 23 000g at 4 °C for 30 min. The precipitate was dissolved in 0.1 mL of deionized water and added 0.1 mL of 50 mM HCl in isopropanol. The mixture was added to 1 mL of 0.1% HCl (v/v) in cold acetone (−20 °C) and allowed to stand at −20 °C for 4 h, followed by centrifugation at 3680g for 10 min at 10 °C. The precipitate was washed twice with cold acetone (0.5 mL) and once with ethyl acetate (0.5 mL), followed by centrifugation at 3680g for 10 min at 10 °C for each washing step. The precipitated protein was dissolved in deionized water (0.5 mL) for further analysis. The concentration of globin was determined by fluorescence excited at 280 nm and emitted at 353 nm as reported.29 Isolation of Human Globin from Whole Blood. Ten microliters of blood was collected freshly and added to a tube containing 10% (v/v) citrate-dextrose solution (the anticoagulant) and centrifuged at 800g at 10 °C for 10 min to separate red blood cells from serum. Globin was isolated after lysis of the red blood cells as reported previously.30 The isolated globin was dissolved in water and quantified by interpolation to a calibration curve constructed from solutions containing various
Table 1. Extraction Efficiency of Hemoglobin from Dried Blood Spots volume of blood on DBSs 25 μL
globin concentration (μg/μL, n = 3) extraction efficiency (% RSD, n = 3)
12.5 μL
6.3 μL
3.1 μL
1/2 cut
1/4 cut
1/8 cut
1/16 cut
9.88 43% (5.3)
4.26 45% (7.0)
1.99 42% (25)
0.68 29% (32)
Scheme 1. Work Flow to Study the Stability of Modifications on Human Hemoglobin Extracted from Dried Blood Spot Cards Stored at 4 °C and at Room Temperature
EXPERIMENTAL SECTION
Materials. Trypsin was from Promega Corporation (Madison, WI). SDS and acetonitrile were purchased from J. T. Baker (Phillipsburg, NJ). The anticoagulant sterile citrate-dextrose solution was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). PerkinElmer 226 spot saver cards were purchased from PerkinElmer Life Sciences (Boston, MA). All reagents are of reagent grade or above. Blotting and Storage of Dried Blood Spots. (A) Fifty microliters of freshly drawn venous human blood was blotted onto a PerkinElmer 226 spot saver card. The DBS card was air-dried at room temperature for 2 h, kept in a plastic bag containing ca. 35 g of anhydrous granular calcium sulfate with a moisture indicator, and stored at 4 °C or room temperature for 1, 7, 14, 21, 28, 35, and 42 days. (B) The DBS card containing 50 μg of blood was air-dried at room temperature for 2 h, followed by drying in a desiccator with vacuum overnight. It was then kept in a vacuum-sealed plastic bag containing a drying agent as described in (A), and stored at −20 °C, 4 °C, or room temperature for for 1, 14, 28, 42, and 56 days. B
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Figure 1. Relative extent of modification on hemoglobin extracted from a dried blood spot stored at 4 °C and at room temperature. (A) Nitrosylation and nitration at tyrosine and (B) oxidation of cysteine. (Magic C18, 5 μm, 100 Å, Michrom BioResource, Auburn, CA) connected to a C18 tip column (75 μm × 110 mm) packed in-house with Magic C18AQ (5 μm, 200 Å, Michrom BioResource). The mobile phases A and B were 5% and 80% acetonitrile (v/v) in 0.1% formic acid (v/v, pH 2.6), respectively. The column was eluted with 4% B (v/v) for 2 min, a linear gradient from 4% B (v/v) to 40% B (v/v) in the next 38 min, then from 40% B (v/v) to 90% B (v/v) in the next 20 min, and maintained at 90% B (v/v) for another 10 min. The conditions were equilibrated with 4% B (v/v) for 20 min before the next run. The flow rate was 300 nL/min. The column was coupled to an LTQ linear ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) fitted with a nanospray ionization (NSI) source. The conditions of mass spectrometry follow the previously described method.27 Relative Extents of Modifications. The selected reaction monitoring (SRM) experiments for peptides containing tyrosine, cysteine, methionine, and these 11 modifications and the corresponding unmodified peptides were performed using the transitions listed in
concentrations of standard hHb in 50 mM HCl and measured by fluorescence as reported.29,31 Globin Digestion with Trypsin. Typically, 50 μg of globin was reconstituted in 100 mM of ammonium bicarbonate (pH 8.0) with 1% SDS (w/v) (total volume 100 μL) and incubated at 95 °C for 10 min. Denatured globin was precipitated with cold acetone (900 μL) after leaving at −20 °C for 15 min, followed by centrifugation at 23 000g for 20 min to discard the SDS-containing supernatant. The precipitate was dissolved in ammonium bicarbonate (100 mM, pH 8.0), added trypsin (5 μg), and incubated at 37 °C for 18 h. After the reaction was stopped by the addition of 0.1% trifluoroacetic acid (v/v, 50 μL), the solution went through a Nylon syringe filter (0.22 μm) before nanoLC−NSI/ MS/MS described below. NanoLC−NSI/MS/MS. Four microliters of each sample (equivalent to 2 μg of globin) was analyzed by an LC system with an UltiMate 3000 RSLCnano system (Dionex, Amsterdam, Netherlands) and a reversed phase C18 precolumn (100 μm × 20 mm) packed in-house C
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Scheme 2. Stability of Modifications on Globin Stored in Solution and Extracted from Dried Blood Spots Stored at Various Temperatures
Table S1 (Supporting Information). The extent of modification on a specific peptide was calculated as the peak area ratio of the modified peptide versus the sum of the peak areas of the modified peptide and its corresponding reference (unmodified) peptide in the SRM chromatograms.27
individuals and the consistency in extraction efficiency of globin from the DBS spot, 1/4 of a spot was used for each triplicate experiment in this study. The purity of globin isolated by this extraction method was reported to be about 96%,18 which is proven to be suitable for quantification of the extent of multiple PTMs by nanoLC-NSI/MS/MS analysis in this study. Measuring the Relative Extents of Modification of Globin by nanoLC−NSI/MS/MS. A total of 11 modifications were examined, including nitration on three tyrosine (α-Tyr-24, α-Tyr-42, and β-Tyr-130) residues, nitrosylation on α-Tyr-24, oxidation of all three methionine (α-Met-32, α-Met-76, and β-Met-55) residues as the sulfoxide, oxidation of all three cysteine (α-Cys-104, β-Cys-93, and β-Cys-112) residues as the sulfonic acid, and α-Cys-104 as the sulfinic acid. The collisioninduced dissociation (CID) mass spectra of these modified peptides are shown in Figure S1 (Supporting Information). After extraction of globin from the DBS card and the subsequent digestion into peptides, the relative extent of modification in globin was analyzed by nanoLC-NSI/MS/MS using the optimal SRM conditions reported27 and quantified by the peak area ratio of the modified peptide versus the sum of the peak areas of the reference peptide and the modified peptide, using the “native reference peptide” method. This relative quantification method allows comparison of the extent of modifications in a protein between samples, but it does not provide the absolute extent of modification. The representative nanoLC-NSI/MS/MS chromatograms are shown in Figure S2 (Supporting Information). The dose-dependent formation of these modifications is shown in Figure S3 (Supporting Information). As we reported recently, the sample pretreatment procedures (such as denaturation at 95 °C) and the electrospray process can introduce artifactual oxidation, such as methionine sulfoxide.25 Experiments with the addition of stable-isotope labeled methionine-containing peptide suggest that artifactual formation of methionine sulfoxide accounts for a small portion of the overall methionine sulfoxide. Stability of Hemoglobin Modifications Stored under Different Conditions. In order to examine the stability of PTMs in human hemoglobin on the DBS, the blood was blotted on the DBS card and dried in air, and the card was sealed in a plastic bag containing drying agent and stored at room temperature (RT) and at 4 °C (Scheme 1). The extents of
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RESULTS AND DISCUSSION Extraction Efficiency of Globin from DBS. Hemoglobin was dissolved in water from the DBS cards, and the cell debris and other water-insoluble ingredients were removed by a syringe filter. A suitable percentage of ethanol was added to precipitate hemoglobin selectively. Sequential wash/precipitation steps were performed to remove salt, metabolites, and other soluble proteins. Using the procedures modified from Funk et al.,18 the protein extracted from the DBS cards was globin with the heme moiety detached because it was exposed to an HCl-containing solvent. A typical 13 mm dried blood spot can encompass about 50 μL of human blood. Assuming a hemoglobin concentration of an individual is 100 mg/mL,32 one DBS contains about 5 mg of hemoglobin. However, most assays for protein adducts typically require between 1 and 20 mg of globin (from Hb).18 Because our nanoLC-NSI/MS/MS is highly sensitive, we examine the feasibility of using a smaller amount of globin from the DBS card. If successful, one spot can be used for multiple assays. After blotting and air-drying, one spot was cut into 1/2, 1/4, 1/8, and 1/16, which is equivalent to 25, 12.5, 6.3, and 3.1 μL of blood, respectively. The extraction efficiency of globin from 1/2 to 1/8 of a spot was ca. 42% to 45%, while that from 1/16 of a spot was 29% compared to the amount of globin extracted from the red blood cells of the same source. The lower extraction efficiency was due to the difficulty in precipitating globin in a small amount and the loss of globin in the washing steps. Funk and co-workers estimated an extraction efficiency of greater than 80%, based on the reported Hb contents in whole blood,18 not by analyzing Hb from the same source of blood. As shown in Table 1, the amount of globin obtained from the smallest portion (1/16) of a DBS spot was enough for the nanoLC-NSI/MS/MS analysis, which used 50 μg of globin for trypsin digestion and an injected equivalent of 2 μg of hydrolysate into the nanoLC-NSI/MS/MS system.27 Considering the lower range of hemoglobin contents in the blood of D
DOI: 10.1021/acs.chemrestox.6b00334 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Figure 2. Extent of modifications of (A) oxidation of methionine, (B) oxidation of β-Cys-93, and (C) nitrosylation at α-Tyr-24 in human hemoglobin extracted from DBS cards kept in vacuum-sealed bags and stored at −20, 4 °C and room temperature (RT) as well as extracted from fresh blood and stored as an aqueous solution at −20 °C.
compared with that in globin extracted directly from blood and stored in aqueous solution at −20 °C from the same source of blood (Scheme 2). The extents of all the modifications were similar in all conditions examined, such as oxidation of methionine residues, (Figure 2A and Figure S5, Supporting Information) except sulfonic acid formation of β-Cys-93, which increases with time and temperature significantly (Figure 2B). Also observed was a slight increase in nitrosylation of α-Tyr-24 (Figure 2C). These results are consistent in four different sample sources. All these 11 PTMs are stable for 14 days at all storage conditions. Furthermore, PTMs in Hb obtained from direct extraction from blood and stored as the aqueous solution at −20 °C are stable for at least 8 weeks. It might be due to the loss of heme group from hemoglobin during the isolation step. Hemoglobin and other hemoproteins can catalyze tyrosine nitration by the H2O2-dependent oxidation of nitrite via the FeIVO intermediate.26,33 The heme group also induces oxidative modifications to hemoglobin during storage of red blood cells for clinical use.34 Without the heme, the oxidationinduced modifications on globin are stable in solution for
modifications in globin from DBSs were measured every week for 6 weeks. Starting from the fourth week, the extents of Tyr-24 nitrosylation and nitration in globin from DBS rose when stored at room temperature, while cysteine oxidation increased after 3 weeks (Figure 1). On the other hand, the extents of oxidation at the three methionine residues were not significantly different, possibly due to their already high levels (Figure S4, Supporting Information). The PTMs measured in this study are associated with oxidative stress. Therefore, it is reasonable to assume that oxygen plays an important role in increasing the extent of modifications. In the next experiment, blood was spotted on the DBS cards in the same manner as that described above, but efforts were made to remove oxygen from the atmosphere of storage of the DBS cards. After the blotted blood was air-dried, the DBS card was dried in a desiccator under vacuum, placed in a vacuum-sealed bag in the presence of drying agent, and stored at −20 °C, 4 °C, and room temperature. Using nanaLC−NSI/ MS/MS, the extent of modifications on globin from the DBS was measured every 2 weeks for up to 8 weeks, and it was E
DOI: 10.1021/acs.chemrestox.6b00334 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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approximately two months, which makes it convenient and feasible from an analytical point of view. Application. The results of these two experiments suggest that oxygen deprivation increases the stability of these PTMs but that it also decreases the convenience of using DBS cards. The most practical way is to handle and store the DBS card in ambient conditions (in air at room temperature) within 2 weeks before measuring these oxidative modifications. This is valuable for studies involving the general population because no special attention is needed for handling and transporting the DBS cards, as long as it is within 2 weeks. After receiving the DBS cards from the subjects, the globin is extracted and can be stored in the freezer for 8 weeks. Only 1/4 of a spot is needed for this analysis, suggesting that multiple assays can be employed for one spot. Hemolysis can take place in vivo with certain diseases or in vitro during blood collection and handling.35 Isolation of globin form DBS cards is particularly suitable for hemolyzed blood samples which are difficult to obtain from intact red blood cells for globin isolation using the conventional method. Once globin is extracted and stored as the aqueous solution in a −20 °C freezer, it is stable for 8 weeks before analysis of these PTMs by nanoLC-NSI/MS/MS. We previously showed that the extents of nitration at α-Tyr24 and α-Tyr-42 of globin are significantly higher in smokers than in nonsmokers and that they are correlated with cigarette smoking.27 We also showed that oxidation of methionine is correlated with diabetes mellitus, age, and the body-mass index.25 Measuring the extents of these PTMs offers a potential biomarker of endogenous oxidative stress, which is associated with diseases including, cancer, diabetes, and inflammatory and cardiovascular diseases.20,22,23 There are advantages of measuring PTMs on hemoglobin. First, hemoglobin is readily available in large quantities. Second, there is no repair of the modifications. Third, hemoglobin has a relatively long lifetime (approximately 120 days) in humans, which allows for monitoring the in vivo status for longer periods of time compared to that of serum albumin (half-life of approximately 20 days).9 Additional benefits concluded from this study include (1) the ease of obtaining heme-free globin in a pure form and in sufficient quantity from DBSs for measuring the extents of modifications by LC−MS/MS and (2) the stability of these oxidative PTMs being 2 weeks on DBSs at ambient conditions and being at least 8 weeks as heme-free globin in aqueous solution at −20 °C. The results should be valuable in using these PTMs as biomarkers for oxidative stress-associated diseases.
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Hauh-Jyun Candy Chen: 0000-0003-1869-4779 Funding
This work was supported by Ministry of Science and Technology of Taiwan (Grants NSC-100-2113-M-194-002MY3 and MOST 103-2113-M-194-003-MY3) and by National Chung Cheng University (to H.-J.C.C.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Mr. Yen-Chen Lee for technical assistance. ABBREVIATIONS DBS, dry blood spot; Hb, hemoglobin; nanoLC−NSI/MS/MS, nanoflow liquid chromatography−nanospray ionization tandem mass spectrometry; PTMs, post-translational modifications; SA, serum albumin; SRM, selected reaction monitoring
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(1) Guthrie, R., and Susi, A. (1963) A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants. Pediatrics 32, 338−343. (2) Wilcken, B., and Wiley, V. (2008) Newborn screening. Pathology 40, 104−115. (3) Parker, S. P., and Cubitt, W. D. (1999) The use of the dried blood spot sample in epidemiological studies. J. Clin. Pathol. 52, 633− 639. (4) McDade, T. W., Williams, S., and Snodgrass, J. J. (2007) What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 44, 899−925. (5) Deglon, J., Thomas, A., Mangin, P., and Staub, C. (2012) Direct analysis of dried blood spots coupled with mass spectrometry: concepts and biomedical applications. Anal. Bioanal. Chem. 402, 2485−2498. (6) Li, F., and Ploch, S. (2012) Will ’green’ aspects of dried blood spot sampling accelerate its implementation and acceptance in the pharmaceutical industry? Bioanalysis 4, 1259−1261. (7) Li, W., and Tse, F. L. (2010) Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules. Biomed. Chromatogr. 24, 49−65. (8) Demirev, P. A. (2013) Dried blood spots: analysis and applications. Anal. Chem. 85, 779−789. (9) Tornqvist, M., Fred, C., Haglund, J., Helleberg, H., Paulsson, B., and Rydberg, P. (2002) Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 778, 279−308. (10) Dingley, K. H., Curtis, K. D., Nowell, S., Felton, J. S., Lang, N. P., and Turteltaub, K. W. (1999) DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Epidemiol. Biomarkers Prev. 8, 507−512. (11) Walker, V. E., Fennell, T. R., Upton, P. B., MacNeela, J. P., and Swenberg, J. A. (1993) Molecular dosimetry of DNA and hemoglobin adducts in mice and rats exposed to ethylene oxide. Environ. Health Perspect. 99, 11−17. (12) Murphy, S. E., Palomino, A., Hecht, S. S., and Hoffmann, D. (1990) Dose-response study of DNA and hemoglobin adduct formation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats. Cancer Res. 50, 5446−5452. (13) Skipper, P. L., and Tannenbaum, S. R. (1990) Protein adducts in the molecular dosimetry of chemical carcinogens. Carcinogenesis 11, 507−518.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00334. List of selected reaction monitoring transitions for measuring the extent of modifications of human hemoglobin; collision-induced dissociation spectra; nanoLC−NSI/MS/MS chromatograms; dose dependency; relative extent of methionine oxidation; and extent of modification in hemoglobin (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*Phone: 886-5-272-9176. Fax: 886-5-272-1040. E-mail:
[email protected]. F
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DOI: 10.1021/acs.chemrestox.6b00334 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
