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Age-Associated Methylation in Human Hemoglobin and its Stability on the Dried Blood Spots as Analyzed by Nanoflow Liquid Chromatography Tandem Mass Spectrometry Hauh-Jyun Candy Chen, and Sun Wai Ip Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00224 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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Chemical Research in Toxicology

Age-Associated Methylation in Human Hemoglobin and its Stability on the Dried Blood Spots as Analyzed by Nanoflow Liquid Chromatography Tandem Mass Spectrometry

Hauh-Jyun Candy Chen* and Sun Wai Ip

Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan

*To

whom

correspondence

should

be

addressed.

Phone:

886-5-272-9176.

886-5-272-1040. E-mail: [email protected].

Keywords: age, dried blood spot, human hemoglobin, methylation, nanoLCMS/MS

1

ACS Paragon Plus Environment

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Abstract Methylation of biomolecules is involved in many important biological processes. The contributing methylating agents arise from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as molecular dosimeter for monitoring chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin

by

nanoflow

liquid

chromatography

tandem

mass

spectrometry

(nanoLCMS/MS) under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in hemoglobin were quantified in nonsmoking subjects with various ages in this study. Among the nine methylation sites, we found that the extents of methylation were significantly higher in elder subjects at the N-terminal and His-20 of -globin, and at the N-terminal and Glu-26 of -globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. We also examined the stability of methylated and ethylated hemoglobin when stored on the dried blood spot cards. The extents of these modifications on hemoglobin are stable for at least 4 weeks stored at room temperature. Our results suggest that age should be considered as a factor when measuring hemoglobin methylation and that dried blood spot is a valuable biomonitoring technique for hemoglobin modifications in epidemiological studies. 3



Introduction Humans are constantly exposed to chemically reactive compounds and the extent of exposure in tissues can be monitored through their reaction products (adducts) with biomolecules, such as DNA and proteins. For the purpose of biomonitoring, blood proteins have been preferred to DNA due to its high abundance and the well-defined life span.1 We focus on hemoglobin adducts because human hemoglobin is abundant, easily obtainable, minimally invasive, and relatively long-lived (ca. 120 days),1 which provides an opportunity for the adducts to accumulate without being repaired. Although DNA damage is directly implicated in the carcinogenesis processes, levels of DNA adducts are much lower than those of protein adducts.1 It might be partly due to the fact that DNA is in the nucleus of a celland protected by histone proteins. Most importantly, cells have developed sophisticated DNA repair mechanism. In addition, linear doseresponse curves are documented for both DNA and Hb adducts in animals and in humans exposed to the same carcinogens or reactive metabolites.2-4 Therefore, hemoglobin adducts can potentially serve as a surrogate for DNA adducts in molecular dosimetry to monitor chemical exposure.5, 6 Methylation plays a central role in many important biological processes, including regulation of transcription, signal transduction, etc.7-10 S-Adenosylmethionine (SAM) is the major endogenous methyl donor for many endogenous processes11 and considering its high concentration in erythrocytes12 suggests it may be in part contributes to methylation of 4


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hemoglobin. In addition, humans are exposed to numerous exogenous methylating agents that are known to methylate DNA and proteins. 13-16 Exogenous methyl donors include those from the environment and cigarette smoke, such as the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

nitrosamine

(NNK) and N-nitrosodimethylamine.17

In our earlier report, the effect of cigarette smoking on the extent of methylation and ethylation at certain sites of hemoglobin was investigated.18 The results showed that the extent of ethylation at six sites of hemoglobin was significantly higher in smokers than in nonsmokers and they correlated with the number of cigarettes smoked per day. On the other hand, no significant correlation was found between smoking and the methylated peptides in hemoglobin,18 which is in agreement with the reports by the research groups of Törnqvist and Hecht.11, 19, 20 During the course of that study, we realized that age seemed to have certain effect on the extent of methylation. We concluded that study with age-matched young subjects to investigate the effect of smoking.18 Subsequently, in this study, we examine the effect of age on hemoglobin methylation in nonsmokers. The contribution of ethylation on DNA and proteins from cigarette smoking has been well documented in the literature.18,

19, 21-27

The detection of small amount of ethylated

adducts in nonsmokers could originate from second- or third-hand smoke or the environment. However, the possibility of endogenous source of ethylation is not known. If there is an association between age and the extent of ethylation, it implies that endogenous ethylating 5



agent(s) exist(s). Thus, ethylation of hemoglobin is included in the same assay as methylation.

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MATERIALS AND METHODS Materials. Human hemoglobin (Hb), dithiothreitol (DTT), and iodoacetamide (IAM) were acquired from Sigma Chemical Co. (St. Louis, MO). Sequencing grade modified trypsin was purchased from Promega Corporation (Madison, WI). PerkinElmer 226 spot saver cards were from PerkinElmer Life Sciences (Boston, MA) and were used for Dried Blood Spots (DBS). All reagents are of reagent grade or above. Study Subjects. The recruitment of volunteers for this study was permitted by the Institutional Review Boards (IRB) of the National Chung Cheng University (IRB no. 100112902) and Kaoshiung Medical University Chung-Ho Memorial Hospital (IRN no. KMUHIRB-E(I)-20170128).

The subjects were all nonsmokers. The mean (± SD) age of

the elder group (8 male and 12 female) was 55.1 ± 9.8 years (ranged 3366 years), while that for the young group (7 male and 7 female) was 23.9  1.8 years (range 2229 years). The mean ( SD) age of the 34 nonsmokers was 42.3  17.3 years (ranged 2266). Globin Isolation from Whole Blood. After blood (0.1 mL) of the subjects was freshly collected in a tube containing the anticoagulant (10% (v/v) of citrate-dextrose solution), it was centrifuged at 800g for 10 min at 10 C to obtain red blood cells, which was washed and lyzed to release globin by following the reported procedures.28 Globin Extraction from Dried Blood Spots. After a DBS card filled with 50 L of fresh blood was air-dried at room temperature for 2 h, it was kept in a plastic bag containing drying agent, and stored at room temperature for 1, 7, 14, 21, 28, 35, 42, and 56 days. Globin 7



was extracted by aqueous ethanol as described.29 Quantification of Globin. The isolated globin was quantified by intrapolation into a calibration curve generated from solutions of standard human Hb by measuring the fluorescence intensity obtained from being excited at 280 nm and emitted at 353 nm.28, 30 Trypsin Digestion of Globin. An aliquot equivalent to 50 μg of Hb was denatured, alkylated by iodoacetamide, and incubated with trypsin (4 μg) at 37C for 18 h as previously described.18 Triplicated experiments were accomplished for each sample. nanoLCNSI/MS/MS Analysis. Four microliters of the trypsin digest was injected onto a nanoflow LC system (UltiMate 3000, Dionex, Amsterdam, Netherlands) coupled to an LTQ linear ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) interfaced with a nanospray ionization (NSI) source. The nanoLC system constituted a C18 precolumn (100

m × 20 mm) packed with Magic C18 (5 m, 100 Å , Michrom BioResource, Auburn, CA) eluting with 0.1% trifuoroacetic acid at a flow rate of 5 L/min for 4.5 min, which was connected to a C18 tip column (75 m 

10 mm) packed in-house with Magic C18AQ (5

m, 200 Å , Michrom BioResource, Auburn, CA). The elution system and MS settings followed the reported conditions.18 The Relative Extents of Methylated and Ethylatied Peptides in Tryptic Digestof Human Globin. The selected reaction monitoring (SRM) experiment was performed by choosing the precursor ion of each peptide and obtaining its product ion scan spectrum in the 8


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nanoLCNSI/MS/MS system described above. The formation of a specific fragment ion from each precursor ion was used to construct the SRM chromatogram. The specific SRM transitions for quantification of modified peptides and their reference peptides followed those reported.18 The extent of modification of a peptide is deliberate as the peak area ratio of the modified peptide versus the sum of the peak areas of the modified peptide and its corresponding unmodified reference peptide in the quantitative SRM chromatograms. Ions below the detection limit in the quantifier SRM chromatogram were considered as zero. For each sample, the experiments were performed in triplicates. Statistical Analysis. GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, www.graphpad.com) was used for statistical analysis. Comparison of the extents of modification between the elder and young groups was achieved by the nonparametric MannWhitney U-test. The relationship between the extents of modification and age or the body mass index (BMI) was examined by the nonparametric Spearman correlation analysis.

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Results and Discussion In this study, we analyzed methylated and ethylated peptides in the trypsin digested peptides of hemoglobin using the relative quantification method developed and reported in our previous report.18 The concept is based on the native reference peptide (NRP) method,31 in which the reference peptide is a native peptide present in the trypsin digest of the protein which elutes closely to the modified peptide. As a result, the method allows correction for variations in protein amounts and peptide recovery in the digestion procedures and it permits comparison of the relative extent of modification between samples, but it does not provide absolute quantitation. Typically, globin isolated from blood of the study subjects was digested with trypsin to give unmodified and modified peptides, which were analyzed by a highly sensitive and specific nanoflow liquid chromatographynanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) system using the selected reaction monitoring (SRM) transitions validated previously.18 The relative extent of modification of a modified peptide is calculated as the peak area ratio of the modified peptide versus the sum of the peak areas of the modified peptide and the corresponding reference peptide in the quantitative SRM chromatograms. The Extents of Methylation and Ethylation in Globin Isolated from Blood and Statistical Significance. In the previous study of the smoking effect, the age in the smoking and nonsmoking groups averaged ( SD) 23.2 ( 1.9) and 23.5 ( 1.1), respectively.18 In this 10


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study, the subjects recruited are all nonsmokers to exclude the effect of smoking. Using the SRM transitions previously reported,18 a total of 9 methylation and 11 ethylation sites in 34 nonsmokers aged 22 to 66 were quantified (Table S1, Supporting Information). The reproducibility of the measurement was indicated by the relative standard deviation (RSD) of the extent of modification in these samples analyzed in triplicates, which averaged 17% for methylated peptides and 19% for ethylated peptides. The subjects were categorized into the elder (age 3466) and the young (2229) groups. Based on the nonparametric Mann-Whitney U-test, the extent of methylation at the N-terminal valine of - and -globin, -His-20, and -Glu-26 were significantly higher in the elder group (n = 20) than the young group (n = 14) with p values of < 0.05 (Table 1 and Figure 1). Among these four sites of modification, methylation at the N-terminal valine of -globin is the site with the most significant change based on the smallest p value (< 0.0001) in the Mann-Whitney U-test. The mean extent of methylation in the elder group, 2.12E-04 (± 6.55E-05), was significantly higher than in the young group, 9.74E-05 (± 5.06E-05). The representative SRM chromatograms of these four methylated peptides and their reference peptides from a sample are shown in Figure 2. In addition, the extents of methylation at these four sites were significantly associated with age as analyzed by the nonparametric Spearman correlation (Figure 3). The most significant correlation was at the N-terminal valine of -globin, with the Spearman correlation coefficient of 0.6049 (p = 11



0.0002). On the other hand, no statistical significance was observed for any ethylated peptides with age, and the possible role of endogenous ethylating agent(s) is excluded. In addition, there is no significant effect on the extents of methylation and ethylation with gender, consistent with the report of no significant association of either adduct with age or gender in nonsmokers by Carmella and co-workers.19 However, the discrepancy in the effect of age might be due to the difference in the analytical methods as well as in the study population. In the study by Carmella et al., modified Edman degradation cleaved the methylated and ethylated N-terminal valine residues in both - and -globin, which were analyzed by GC-MS.19 In that study, the age of 29 nonsmokers ranged from 20 to 57 (mean 29.4  9.2).19 In this study, the age of the 34 nonsmokers ranged from 22 to 66 (mean 42.3  17.3). The wider age range in this study might be helpful in examining its effect on the extent of modifications. Stability of Methylation and Ethylation in Hemoglobin Stored on the Dried Blood Spot Card. A fixed volume of blood (50 L) was blotted onto the DBS card, air-dried, and stored in a plastic bag with drying agent at room temperature for up to 56 days (8 weeks). Hemoglobin was extracted from a spot from the DBS card using the optimal concentration of aqueous ethanol.32 During purification of hemoglobin by precipitation with acidic isopropanol, the heme group was detached and globin was obtained in good purity. The extraction efficiency from the DBS card does not vary much with the storage time, with an 12


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average of 74%. The trypsin digestion experiment started with 50 g of globin and was performed in triplicates on samples from three subjects (No. 14, 18e, and 20e). As shown in Figure 4, methylation at His-50 of the -globin and N-terminal Val, Lys-66, His-77, and Cys-93 of the -globin increase after 35 or 42 days in some or all three samples. Taken together, all the methylated peptides are stable on the DBS card for 28 days. In contrast, the extents of ethylation in nearly all the sites were stable throughout the period of 8 weeks (Figure 5). Globin extracted from DBS at day 1 was stored at 20 C for 55 days and the extents of modifications were compared with those at day 56. The results show that the mean RSD of the extent of modification ratio (Day 56 vs. Day 1) at these sites in these samples is 15% (Table S2, Supporting Information), similar to the variation of the assay. Methylation and ethylation on globin are stable in aqueous solution stored at 20 C for the longest period of time examined (8 weeks), same as the stability of nitrative and oxidative modifications under this condition.29 Globin is less prone to oxidation than hemoglobin because the heme prosthetic group is lost during the extraction from blood. Heme and hemoproteins are known to induce nitrative and oxidative modifications on proteins.28, 33-35 Several types of oxidative modifications on hemoglobin arise during routine storage of red blood cells for transfusion.35 The extents of nitration and oxidation on hemoglobin stored on DBS cards were reported to be ca. 2 weeks at room temperature in the air.29 Unlike oxidative modifications 13



which are prone to increase in the presence of oxygen, there are much less methylating and ethylating agents in the ambient air. Proteins can be degraded during storage during storage in the air at room temperature. Because the unmodified globin is much more abundant than modified globin, degradation of unmodified globin can lead to increase in the extent of modifications. The results showing that ethylated peptides are more stable than the methylated peptides led us to assume that there are more methylating agent than ethylating agent in the ambient air because the same unmodified reference peptide is used for quantification of methlation and ethylation at the same site. Our results agree with this assumption and indicate that there are more methylating than ethylating agent in the ambient air, but both are in much lower concentrations than the oxidizing agents. Nonetheless, the possibility of matrix effects produced by alkyl attachment during storage or artifact formation cannot be excluded. Methylation of hemoglobin has been investigated mostly on methylated N-terminal valine,11, 19, 20 while methylation on histidine and cysteine have been identified as well.11 The natural biochemical SAM has been suggested as the major endogenous source of N-terminal methyl valine, considering its high concentration in erythrocytes and its reactivity,12 while methyl histidine and cysteine can be incorporated into hemoglobin during protein biosynthesis.36 The concentration of SAM can be affected by diet, life style, and hereditary factors.13-16 The ratios of SAM versus its demethylated counterpart S-adenosylhomocysteine 14


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(SAH) in red blood cells increase as a function of age.13 Methylation of human lens crystallins, the long-lived proteins, increases with age, which was believed to be mediated by SAM nonenzymatically.37 Other methyl donors include dietary folate, vitamin B6, and B12. However, concentrations of SAM and the ratios of SAM vs. SAH do not change with diet. 15 The concentrations of SAM in erythrocytes are gender-dependent,13 but the extent of Hb methylation is not (in this study). Genetic polymorphisms of catechol-O-methyltransferase and methylenetetrahydrofolate reductase are factors that can modify the bioavailability of SAM.38, 39 However, there is no conclusive evidence on the association of these two enzymes with age. Methylation of DNA primarily takes place at the N7-position of guanine, forming N7-methylguanine (7-MeGua).40, 41 Similar to methylation of proteins, levels of 7-MeGua are also influenced by several factors, including smoking, diet, lifestyle, and age.14,

42

The

background levels of 7-MeGua in tissue DNA of rats and mice increase with age,43, 44 and so does 7-MeGua excretion in human urine.42 From the analytical point of view, measuring methylated and ethylated hemoglobin adducts by our nanoLCNSI/MS/MS assay on the modified peptides has several advantages over the analysis of DNA adducts in urine or tissues. First, hemoglobin is much more abundant and easily obtainable in pure form than DNA. Although urine is noninvasive and attainable, its complex matrix is disadvantageous for the MS-based assay and sample 15



pretreatment is critical for the success of the assy. Second, trypsin digestion is currently a well optimized and routine procedure. It allows analysis of multiple sites on peptides and it is straight forward without derivatization. Third, the assay is very sensitive and specific and it requires very small amount of blood samples which can be obtained from DBS after being stored for ca. one month under ambient condition. Ethylation at several sites of hemoglobin can reflect the exposure to smoke during the 4-month period of an individual.18 Our results conclude that age should be considered as a factor when studying hemoglobin methylation and that DBS is a valuable biosampling technique for hemoglobin methylation and ethylation in studies involving large population.

Acknowledgements. We thank Dr. Deng-Chyang Wu of the Kaoshiung Medical University Chung-Ho Memorial Hospital for providing part of the blood samples.

Funding This work was supported by Ministry of Science and Technology of Taiwan (Grants MOST 103-2113-M-194-003-MY3 and MOST 106-2113-M-194-013-MY3) and National Chung Cheng University (to H.-J.C.C.).

Supporting Information 16


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The Supporting Information is available free of charge on the ACS Publications website. Relative extent of methylated and ethylated peptides in hemoglobin isolated from nonsmoking subjects; Comparison of the extent of methylated and ethylated peptides in hemoglobin isolated from DBS card at day 1 and those stored in aqueous solution at 20 C for 55 days. (PDF)

Abbreviations: DBS, dried blood spot; hHb, human hemoglobin; nanoLCNSI/MS/MS, nanoflow liquid chromatographynanospray ionization tandem mass spectrometry; SRM, selected reaction monitoring;

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Chen, H. J., and Lin, C. R. (2013) Simultaneous quantification of ethylpurine adducts in human urine by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry. J Chromatogr A, 1322, 69-73. Singh, R., Kaur, B., and Farmer, P. B. (2005) Detection of DNA damage derived from a direct acting ethylating agent present in cigarette smoke by use of liquid chromatography-tandem mass spectrometry. Chem. Res. Toxicol., 18, 249-256. Chen, H. J., and Chen, Y. C. (2012) Reactive nitrogen oxide species-induced post-translational modifications in human hemoglobin and the association with cigarette smoking. Anal. Chem., 84, 7881−7890. Chen, H. C., Fan, C. H., and Yang, Y. F. (2016) Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry. Chem. Res. Toxicol., 29, 2157−2163. Boys, B. L., and Konermann, L. (2007) Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system. J. Am. Soc. Mass Spectrom., 18, 8−16. Ruse, C. I., Willard, B., Jin, J. P., Haas, T., Kinter, M., and Bond, M. (2002) Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry. Anal. Chem., 74, 1658−1664. Funk, W. E., Waidyanatha, S., Chaing, S. H., and Rappaport, S. M. (2008) Hemoglobin adducts of benzene oxide in neonatal and adult dried blood spots. Cancer Epidemiol. Biomarkers Prev., 17, 1896−1901. Chen, H. J., Chang, C. M., Lin, W. P., Cheng, D. L., and Leong, M. I. (2008) H2O2/nitrite-induced post-translational modifications of human hemoglobin determined by mass spectrometry: redox regulation of tyrosine nitration and 3-nitrotyrosine reduction by antioxidants. Chembiochem, 9, 312-323. Grzelak, A., Balcerczyk, A., Mateja, A., and Bartosz, G. (2001) Hemoglobin can nitrate itself and other proteins. Bba-Gen Subjects, 1528, 97−100. Wither, M., Dzieciatkowska, M., Nemkov, T., Strop, P., D'Alessandro, A., and Hansen, K. C. (2016) Hemoglobin oxidation at functional amino acid residues during routine storage of red blood cells. Transfusion, 56, 421−426. Kautiainen, A., Osterman-Golkar, S., and Ehrenberg, L. (1986) Misincorporation of alkylated amino acids into hemoglobin--a possible source of background alkylations. Acta Chem Scand B, 40, 453-456. Truscott, R. J., Mizdrak, J., Friedrich, M. G., Hooi, M. Y., Lyons, B., Jamie, J. F., Davies, M. J., Wilmarth, P. A., and David, L. L. (2012) Is protein methylation in the human lens 20


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a result of non-enzymatic methylation by S-adenosylmethionine? Exp Eye Res, 99, (38)

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Table 1. Relative Extent of Methylation and Ethylation in Hemoglobin Isolated from Nonsmoking Subjects and Statistical Analysis

relative extent of modification (mean ± SD) elder nonsmokers (n=20)

Mann-Whitney Spearman correlation

young nonsmokers (n=14)

U-test

coefficient (p value)b

p valuea

age

α-1VMe

6.95E-04 ± 6.66E-04

2.66E-04 ± 8.97E-05

0.0082

0.4640 (0.0057)

Me

α- H

1.87E-03 ± 1.02E-03

1.19E-03 ± 6.95E-04

0.0263

0.3601 (0.0365)

α-50HMe

2.58E-04 ± 2.58E-04

2.86E-04 ± 1.14E-04

α- H

7.99E-05 ± 1.19E-04

3.54E-05 ± 5.07E-05

β-1VMe

2.12E-04 ± 6.55E-05

9.74E-05 ± 5.06E-05

< 0.0001

0.6049 (0.0002)

26 Me

β- E

3.44E-02 ± 1.93E-02

1.97E-02 ± 1.31E-02

0.0219

0.3703 (0.0311)

β-66KMe

1.03E-04 ± 4.80E-05

1.20E-04 ± 1.17E-04

Me

β- H

1.85E-03 ± 2.08E-03

9.30E-04 ± 1.88E-04

β-93CMe

1.72E-04 ± 7.08E-05

1.72E-04 ± 9.14E-05

α-1VEt

8.35E-05 ± 8.61E-05

1.07E-04 ± 1.04E-04

α-16KEt

6.25E-05 ± 5.05E-05

1.03E-04 ± 7.42E-05

α- H

Et

1.64E-05 ± 1.38E-05

3.79E-05 ± 4.96E-05

α-72HEt

3.40E-06 ± 4.24E-06

3.91E-06 ± 4.65E-06

α- H

2.55E-05 ± 1.88E-05

3.18E-05 ± 2.11E-05

β-1VEt

2.11E-05 ± 1.44E-05

2.05E-05 ± 1.53E-05

β- K

Et

3.60E-05 ± 6.19E-05

1.01E-04 ± 9.85E-05

β-66KEt

1.20E-05 ± 1.30E-05

2.64E-05 ± 2.70E-05

Et

β- H

4.08E-06 ± 2.74E-06

4.16E-06 ± 3.17E-06

β-92HEt

3.64E-05 ± 3.27E-05

3.17E-05 ± 2.08E-05

β- C

8.82E-05 ± 1.40E-04

1.25E-04 ± 2.56E-04

20

72

Me

77

50

87

17

77

93

Et

Et

a

The two-tailed p value between elder nonsmokers (n=20) and young nonsmokers (n=14) using the

nonparametric Mann−Whitney U-test. The two-tailed p value was shown in parentheses. b

The Spearman correlation coefficient between the extent of modification and age of the subjects

(n=34).

22


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Figure Legends. Figure 1. nanoLCNSI/MS/MS chromatograms of methylated peptides (A) 1VMeLSPADK and VGA20HMeAGEYGAEALER with their reference peptide in -globin and (B) 1

VMeHLTPEEK and VNVDEVGG26EMeALGR with their reference peptide in -globin under

the SRM mode (sample No. 18e). Multiple SRM chromatograms of the modified peptides are shown on the right panel with the SRM used for quantification marked in red rectangle. Figure 2. Statistical significance in the extents of methylation at (A) N-terminal of -globin, (B) -His-20, (C) N-terminal of -globin, and (D) -Glu-26 between elder and young subject groups. Figure 3. Correlation between age and the extents of methylation at (A) N-terminal of -globin, (B) -His-20, (C) N-terminal of -globin, and (D) -Glu-26. Figure 4. Extent of methylation on hemoglobin extracted from the dried blood spots stored at room temperature for 56 days. Figure 5. Extent of ethylation on hemoglobin extracted from the dried blood spots stored at room temperature for 56 days.

23



Figure 1. (A)

(B)

24


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Figure 2. (A)

(C)

(B)

(D)

25



Figure 3. (A)

(B)

(C)

(D)

26


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Figure 4.

27



Figure 5.

28


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Age-Associated Methylation in Human ... - ACS Publications

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