Article Cite This: Chem. Res. Toxicol. 2017, 30, 2074-2083
pubs.acs.org/crt
Simultaneous Mass Spectrometric Analysis of Methylated and Ethylated Peptides in Human Hemoglobin: Correlation with Cigarette Smoking Hauh-Jyun Candy Chen,* Sun Wai Ip, and Fu-Di Lin Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan S Supporting Information *
ABSTRACT: 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-His87, β-terminal Val, β-His-77, and β-Cys-93 in Hb were significantly higher in smokers than in nonsmokers (p < 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.
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INTRODUCTION The majority of the exogenous mutagens contained in the environment need to be metabolically activated to form the ultimate reactive species that bind with biomolecules, such as proteins and nucleic acids. In the case of tobacco-specific nitrosamines and polycyclic aromatic hydrocarbons, they become potent alkylating agents after being metabolized by cytochrome P450 enzymes and react with DNA forming mutagenic DNA adducts.1,2 Nonetheless, the lack of the effect of genetic polymorphisms of the CYP1A1, GSTM1, GSTP1, GSTT1, GSTP1, or NQO1 on the risk of lung cancer in smokers suggests that the direct-acting genotoxic agents play an important role in smoking-related lung cancer.3,4 Furthermore, Singh and co-workers demonstrated that smoke generated from cigarettes contains a structurally unidentified direct-acting ethylating agent that reacts with DNA to form N7-ethylguanine.5 While DNA adducts are used as biomarkers for assessing cancer risk by mutagens, protein adducts derived from xenobiotic carcinogens and endogenous reactive species or their metabolites can cause pathological conditions or diseases. Proteins are more susceptible to react with the reactive species than DNA because © 2017 American Chemical Society
DNA is sheltered in the nucleus (except mitochondrial DNA). Therefore, protein adducts are generally present in higher levels than DNA adducts. A dose-responsive increase in both hemoglobin (Hb) and DNA adducts was observed in ethylene oxide-exposed animals, suggesting that Hb adducts can serve as surrogate biomarkers for DNA adduct in target tissues.6 In blood, Hb and serum albumin (SA) are much more abundant than DNA.7 It is therefore much easier to obtain large quantities of Hb and SA than DNA from blood. Among the protein adducts in blood, those from Hb and SA have been used as promising biomarkers for exposure to pollutants.7 Human Hb adducts allows us to monitor the internal dose of exposure for a longer period of time than does SA because the life span of human Hb is 126 days, while the half-life of HSA is 20 days.8−10 Mass spectrometry is the method of choice for the analysis of protein adducts. Methylation and ethylation of Hb have been measured as the Edman degradation products at the N-terminal valine using GC-MS or LC−MS/MS.11−15 Zhang and coReceived: August 18, 2017 Published: October 2, 2017 2074
DOI: 10.1021/acs.chemrestox.7b00234 Chem. Res. Toxicol. 2017, 30, 2074−2083
Article
Chemical Research in Toxicology
The peptides were analyzed in the positive ion mode by nanospray ionization with a spray voltage of 1.6 kV. Mass spectrometry was operated in a data-dependent scan mode, in which one full scan with m/ z 300−2000 in the Orbitrap at a resolution of 60,000 at m/z 400 using a rate of 30 ms/scan. The five most intense peaks were selected for fragmentation with a normalized collision energy value of 35% in the LTQ. A repeat duration of 180 s was applied to exclude the same m/z ions from the reselection for fragmentation. Peptide characterization was performed using the in-house MASCOT v2.3.02 search engine on the Swiss-Prot 56 human protein database. Two miscleavage sites were allowed for digestion with trypsin at sites of lysine and arginine. The mass tolerance should be below 5 ppm for precursor ions and below 0.8 Da for product ions. All MS/MS spectra were searched against the database mentioned above for detecting variable modifications, including methylation (+ 14.01535) and ethylation (+ 28.03130) of aspartate, glutamate, cysteine, histidine, lysine, arginine, tryptophan, terminal valine, and carboxyamidomethylation (+57.02146) on cysteine residues, and one missed cleavage on trypsin was allowed. The cutoff score was set at 20 (p < 0.05) to eliminate low score peptides, and only “rank1” (best match for each MS/ MS) peptides were included. nanoLC−NSI/MS/MS System. Four μL of each sample was injected onto a nanoflow LC system (UltiMate 3000, Dionex, Amsterdam, Netherlands) interfaced with an LTQ linear ion trap mass spectrometer (Thermo Electron Corp., San Jose, CA) equipped with a nanospray ionization (NSI) source. A C18 precolumn (100 μm × 20 mm) packed in-house with Magic C18 (5 μm, 100 Å, Michrom BioResource, Auburn, CA) was eluted with 0.1% trifuoroacetic acid at a flow rate of 5 μL/min for 4.5 min. It was connected to a C18 tip column (75 μm × 110 mm) packed in-house (Magic C18AQ, 5 μm, 200 Å, Michrom BioResource, Auburn, CA). The mobile phases A and B were composed of 5% and 80% acetonitrile in 0.1% formic acid (pH 2.6), respectively. The system started elution with 4% B for the first 4.5 min, followed by a linear gradient from 4% B to 40% B in the next 38 min and from 40% B to 90% B in the next 20 min. It was maintained at 90% B for 10 min and equilibrated with 4% B for another 20 min before the next injection. The flow rate was at 300 nL/min. All MS2 experiments for peptide characterization were performed at a heated capillary temperature of 200 °C with a capillary voltage of 13 V, a tube lens voltage of 80 V, a source voltage of 1.6 kV, a source current of 100 μA, a normalized collision energy of 35%, and the ion gauge pressure of 7.2 × 10−6 Torr. Semiquantification of Methylated and Ethylated Peptides in Hemoglobin. The selected reaction monitoring (SRM) experiment of each peptide was performed by selecting the precursor ion and acquiring its product ion scan spectrum in the nanoLC−NSI/MS/MS system. The formation of a specific fragment ion from each precursor ion was used to construct the chromatogram. The specific SRM transitions for modified peptides and their reference peptides are listed in Table 3. The relative extent of modification on a 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 SRM chromatograms. The drop perpendicular method was used for manual integration of chromatographic peaks. The 7-point Gaussian smoothing method was employed for peak detection. The experiments were performed in triplicates for each sample. Isolation and Quantification of Hemoglobin Isolated from Blood. Blood (0.1 mL) was freshly collected in a tube containing the anticoagulant, and it was centrifuged at 800g at 10 °C for 10 min to obtain red blood cells, which was washed and lyzed as reported.17 The globin isolated was quantified by intrapolation into a calibration curve constructed from solutions of standard hHb measuring fluorescence excited at 280 nm and emitted at 353 nm.17,19 Study Subjects. The study subjects were recruited under the approval of the Institutional Review Boards (IRB) of the National Chung Cheng University (IRB no. 100112902) including 13 smokers (all male) and 13 nonsmokers (7 male and 6 female). The mean (±SD) age was 23.2 ± 1.9 and 23.5 ± 1.1 for smokers and nonsmokers, respectively. The average number of cigarettes smoked per day of smokers was 10.2 ± 5.8.
workers used LC-MS/MS to quantify the degree of methylation at the N-terminal of the β-chain in the Hb of rats treated with methyl methanesulfonate (MMS), as the signature octapeptide in trypsin digest.16 These studies only measured the extents of methylation and/or ethylation of N-terminal valine residues from either β-globin or both α- and β-globin. However, Nterminal valine residues are not the only reactive sites of Hb. Other amino acid residues such as lysine, arginine, histidine, aspartate, and glutamate in Hb are also prone to alkylation, but this possibility has not been investigated previously. In this study, a total of 18 methylated and ethylated peptides were identified by a high-resolution mass spectrometer using the shot-gun proteomic approach. These 18 modified peptides were quantified by a highly specific and sensitive nanoflow LC− nanospray ionization tandem mass spectrometry (nanoLC− NSI/MS/MS), which was applied for the analysis of globin isolated from the blood samples of smokers and nonsmokers.
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MATERIALS AND METHODS
Materials. Dithiotreitol (DTT), iodoacetamide (IAM), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and human hemoglobin (hHb) were purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin was acquired from Promega Corporation (Madison, WI). All reagents are of reagent grade or above. Dose-Dependent Formation of Methylated Peptides by MMS. The solution containing 0.1 mM of commercial hHb was incubated with various concentrations of MMS (0, 5, 10, 50, or 200 μM) in potassium phosphate buffer (0.1 M, pH 7.4) at 37 °C for 12 h. The mixture was added ice-cold acetone (10:1, v/v) to precipitate the protein. The solution was allowed to stand at −20 °C for 30 min and centrifuged at 0 °C for 20 min at 23,000g. The supernatant containing excess alkylating reagent was discarded, and the precipitate was air-dried. The extents of methylation were quantified as described below. The experiments were performed in triplicates for each concentration. The dose-dependency was plotted as the extent of methylation versus the MMS concentration. Time-Dependent Formation of Ethylated Peptides by EMS. A solution containing commercial hHb (0.1 mM) was incubated with EMS (10 mM) in potassium phosphate buffer (0.1 M, pH 7.4) at 37 °C for 0, 12, 24, and 48 h. The mixture was processed as described above, and extents of ethylation were quantified as described below. The experiments were performed in triplicates for each time point. The time dependency was plotted as the extent of ethylation versus time of incubation. Enzyme Digestion. The precipitated Hb was dissolved in doubly distilled water and quantified based on the tryptophan-induced fluorescence measuring fluorescence at the excitation wavelength at 280 nm and the emission wavelength at 353 nm.17 An equivalent to 50 μg of Hb was denatured, alkylated, and digested as reported18 except that iodoacetamide was used to replace iodoacetic acid. After the digestion was stopped by trifluoroacetic acid, 4 μL of the entire solution was subjected to UPLC with accurate mass measurement or nanoLC− NSI/MS/MS system described below. Characterization of Sites and Types of Modification by UPLC with Accurate Mass Measurement. The hHb digest described above was analysis by a nanoACOUITY Ultra Performance LC system (Waters Corp., Milford, MA) connected to an LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, CA). Reversed phase nanoLC system was employed on an ACQUITY UPLC Symmetry C18 trap column (180 μm × 20 mm, 5 μm), followed by separation using a ACQUITY UPLC Peptide BEH C18 column (75 μm × 250 mm, 1.7 μm, Waters Corp., Milford, CA). The mobile phases A and B were composed of 0.01% formic acid (pH 3.6) and acetonitrile in 0.01% formic acid, respectively. Both columns were eluted with 10% B for the first 5 min, followed by a linear gradient from 10% B to 35% B in the next 25 min and from 35% B to 90% B in the next 5 min, maintained at 90% B for another 5 min at a flow rate of 300 nL/min. 2075
DOI: 10.1021/acs.chemrestox.7b00234 Chem. Res. Toxicol. 2017, 30, 2074−2083
Article
Chemical Research in Toxicology Scheme 1
Table 1. Characterization of Peptides in Human Hemoglobin Treated with Methyl Methanesulfonate by High-Resolution Mass Spectrometrya AA startend
methylated peptides
Peptide from α-Globin 1 Me V LSPADK 1−7 17−31 VGA20HMeAGEYGAEALER 41−56 TYFPHFDLS50HMeGSAQVK 62−90 VADALTNAVA72HMeVDDMPNALSALSDLHAHK Peptide from β-Globin 1 Me V HLTPEEK 1−8 18−30 VNVDEVGG26EMeALGR 66 Me K VLGAFSDGLAHLDNLK 66−82 67−82 VLGAFSDGLA77HMeLDNLK 83−95 GTFATLSELH93CMeDK a
m/z expt.
z
MW expt.
MW calc.
ΔMW (ppm)
Mascot score
modification
743.4218 772.3713 924.4526 1004.1679
1 2 2 3
742.4218 1542.7425 1846.9052 3009.5037
742.42250 1542.7426 1846.9003 3009.4978
−0.94 −0.06 2.65 1.96
38.64 45.54 50.46 78.76
−H + Me (V, +14) −H + Me (H, +14) −H + Me (H, +14) −H + Me (H, +14)
966.5198 664.8352 906.4977 842.4493 718.3406
1 2 2 2 2
965.5198 1327.6704 1810.9953 1682.8985 1434.6812
965.5182 1327.6736 1810.9941 1682.8985 1434.6813
1.66 −2.11 0.66 −0.42 −0.07
34.33 112.02 40.08 112.61 82.56
−H + Me (V, +14) −H + Me (E, +14) −H + Me (K,+14) −H + Me (H, +14) −H + Me (H, +14)
A solution of hHb (100 μM) was incubated with MMS (1 mM) at 37 °C for 12 h.
Statistical Analysis. Statistical analysis was performed by GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, www.graphpad.com). Comparison of the extents of methylation and ethylation between two subject groups was achieved by the nonparametric Mann−Whitney U-test. The correlation between the extent of methylation or ethylation with the number of cigarettes smoked per day or the body mass index (BMI) was performed by the nonparametric Spearman correlation analysis.
Identification of Methylated and Ethylated Peptides in Hemoglobin. A solution of commercially available hHb was incubated with MMS at 37 °C for 24 h, followed by alkylation and trypsin digestion. The accurate mass of the peptides was measured by a high-resolution mass spectrometer in the datadependent scan mode. The results revealed a total of 7 methylation sites, including the N-terminal of α-globin, α-His20, α-His-50, α-His-72 and N-terminal of β-globin, β-Glu-26, βLys-66, β-His-77, and β-Cys-93. As listed in Table 1, the accurate mass measurement showed a mass increase of 14 Da for these peptides, suggesting the substitution of a methyl group. The Mascot score in peptide identification is inversely related to the probability of matching the MS/MS experimental data of the peptides with sequences found in the database. The Mascot scores were >34, and the mass accuracy was below 3 ppm for the identified methylated peptides, supporting the confidence of the identification. To confirm the sites of Hb ethylation, EMS, a weaker alkylating agent than MMS, was incubated with hHb at 37 °C for various time periods up to 48 h. After digestion with trypsin, accurate mass analysis of the peptides identified 11 ethylation sites, including N-terminal valine, Lys-16, His-50, His-72, and
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RESULTS Because of the low levels of methylation and ethylation in blood Hb, the methylation and ethylation sites were characterized with Hb incubated in vitro with a methylating or ethylating agent, respectively. The accurate masses of peptides from the treated Hb digest were measured, and their identity was confirmed by a sequence database search and based on the MS2 mass spectra from a high-resolution mass spectrometer. The relative extent of modification in each peptide was semiquantified by the SRM transitions using native peptides present in the digest as reference. This SRM method was applied to quantify the relative extent of methylation and ethylation of Hb isolated from human blood (Scheme 1). 2076
DOI: 10.1021/acs.chemrestox.7b00234 Chem. Res. Toxicol. 2017, 30, 2074−2083
Article
Chemical Research in Toxicology
Table 2. Characterization of Peptides in Human Hemoglobin Treated with Ethyl Methanesulfonate by High-Resolution Mass Spectrometrya AA startend
ethylated peptides
Peptide from α-Globin 1 Et 1−7 V LSPADK 12−31 AAWG16KEtVGAHAGEYGAEALER 41−56 TYFPHFDLS50HEtGSAQVK 62−90 VADALTNAVA72HEtVDDMPNALSALSDLHAHK 62−90 VADALTNAVAHVDDMPNALSALSDL87HEtAHK Peptide from β-Globin 1 Et 1−8 V HLTPEEK 9−30 SAVTALWG17KEtVNVDEVGGEALGR 66 Et 66−82 K VLGAFSDGLAHLDNLK 67−82 VLGAFSDGLA77HEtLDNLK 83−95 GTFATLSEL92HEtCCAMDK 83−95 a
GTFATLSELH93CEtDK
m/z expt.
z
MW expt.
MW calc.
ΔMW (ppm)
Mascot score
modification
379.2191 1036.0171 931.4598 1008.8405 1008.8425
2 2 2 3 3
756.4381 2070.0342 1860.9195 3023.5214 3023.5274
756.4381 2070.0282 1860.9177 3023.5174 3023.5174
0.00 2.90 0.97 1.32 3.31
41.05 95.85 72.59 95.37 37.61
−H + Et (V, +28) −H + Et (K, +28) −H + Et (H, +28) −H + Et (H, +28) −H + Et (H, +28)
980.5358 752.7305 913.5059 849.4605 753.8595
1 3 2 2 2
979.5358 2255.1915 1825.0117 1696.9210 1505.7190
979.5338 2255.1910 1825.0098 1696.9148 1505.7184
2.04 0.22 1.04 3.65 0.40
33.74 63.30 88.50 111.34 43.8
725.3556
2
1448.6966
1448.6970
−0.28
87.51
−H + Et (V, +28) −H + Et (K, +28) −H + Et (K, +28) −H + Et (H, +28) −2H + Et (H, +28) + CAM (C, +57) −H + Et (C, +28)
A solution of hHb (100 μM) was incubated with EMS (10 mM) at 37 °C for 24 h. CAM: carboxyamidomethyl (CAM) modification.
His-87 of α-globin and N-terminal, Lys-17, Lys-66, His-77, His92 and Cys-93 of β-globin. In Table 2, the mass increase of the peptides was 28 Da (substitution of an ethyl group), and the mass accuracy for the ethylated peptides was below 4 ppm with their Mascot scores >34. The collision-induced dissociation (CID) spectra of these peptides provided evidence for the sites and types of modification (Figure S1, Supporting Information). Relative Quantification of Methylated and Ethylated Peptides in Hemoglobin. The “native reference peptide (NRP)” method was adopted to quantify the extent of methylation and ethylation in Hb. The NPR method allows for correction of variations in the protein amounts and the recovery of peptides during the enzyme digestion steps, using the peptide present in the protein digest as the reference. This NRP method has been applied for quantification of various post-translation modifications, including phosphorylation, nitration, oxidation, chlorination, glutathionylation, carboxymethylation, and carboxyethylation.17,20−25 In this study, the native peptides present in the globin digest that elute closely with the methylated and ethylated peptides were chosen as the reference. The retention time difference between the modified and reference peptides (ΔRT) were within 5 min (Table 3). The fragment ions chosen in the SRM transitions include the modification sites. To differentiate ethylation at His-72 and His87 on the same peptide VADALTNAVA72HVDDMPNALSALSDL87HAHK of α-globin, fragment ions b15′ (+1) at m/z 1551.77 and y15′ (+2) at m/z 816.94 were used in the SRM transitions for the quantification of His-72 and His-87, respectively. In addition, each modified peptide was monitored by at least two more SRM transitions; the transitions with the highest signals intensity were used as quantifiers, and the others were used as the qualifiers. For ethylation at His-72, the b11′ (+1) ion at m/z 1091.61, b12′ (+1) ion at m/z 1 190.68, b14′ (+1) ion at m/z 1420.73, and b24′ (+3) ion at m/z 807.40 were used as qualifiers. These fragments ions do not include His-87 and should be exclusive for the modification at His-72. For ethylation at His-87, the y5′ (+1) ion at m/z 633.41, y6′ (+1) ion at m/z 748.43, and y8′ (+1) ion at m/z 948.55, which do not include His72, were used as qualifiers. Methylation at His-72 was quantified using b15′ (+1) at m/z 1537.75, and the b11′ (+1) ion at m/z 1077.59 and b14′ (+1) ion at m/z 1406.71 were used as qualifiers. The same fragment ions for the methylated and ethylated
peptides were chosen so that the extent of methylation and ethylation could be compared in the same sample. The relative extent of methylation (or ethylation) was calculated from the peak area of the methylated (or ethylated) peptide versus the sum of the peak areas of the methylated (or ethylated) peptide and its reference peptide under the SRM transitions (Table 3). The appropriate SRM transitions should include the modification sites in the fragment ion for the modified peptide, and the same fragment ion is used for the methylated and ethylated peptides. For instance, the y7′ (+1) ion at m/z 824.48 and m/z 838.50 was used in the SRM transitions for the methylated and ethylated β-His-77-containing peptide, respectively, while the y7 (+1) ion for the unmodified peptide was at m/z 810.45. Dose-Dependent Formation of Methylated Peptides Induced by MMS. Human Hb obtained from a commercial source was incubated with various concentrations of MMS at 37 °C for 12 h to identify the target peak in the LC−MS chromatograms. The extent of methylation of the 9 methylated peptides increased with increasing concentrations of MMS, (Figure S1, Supporting Information), suggesting that methylation at these sites adequately reflects the exposure to MMS. Compared to the corresponding unmodified peptides, methylation at the N-terminal valine, histidine, and glutamate residues led to delayed retention of 1−2 min in the chromatograms (Table 3). Time-Dependent Formation of Ethylated Peptides Induced by EMS. The ethylating agent EMS is a structural analog of MMS with a lower reactivity. In the solution of Hb incubated with EMS, the extent of modification of the ethylated peptides increased in a time-dependent manner (Figure S2), indicating that these ethylated peptides can reveal the exposure to EMS. The peptides ethylated at the N-terminal valine and histidine residues eluted
