Reactive Nitrogen Oxide Species-Induced Post-Translational

Article pubs.acs.org/ac

Reactive Nitrogen Oxide Species-Induced Post-Translational Modifications in Human Hemoglobin and the Association with Cigarette Smoking Hauh-Jyun Candy Chen* and Yu-Chin Chen Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan S Supporting Information *

ABSTRACT: 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 βCys-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. of nitration was estimated to be ∼1 in 106 tyrosines24 and ∼1 in 104 tyrosines under inflammatory conditions.19 Post-translational protein tyrosine nitration mainly causes inhibition of protein functions directly or via inhibition of tyrosine phosphorylation.25−27 Tyrosine nitration is not a permanent modification, and it can be reversed under redox control. We previously reported that 3NT can be reduced to 3aminotyrosine (3AT) by lipoyl dehydrogenase or by hemoproteins in the presence of biological antioxidants, such as dihydrolipoic acid and ascorbate.28,29 Mirzaei and co-workers identified 3NT, along with 3AT, in α-synuclein in cells treated with an inhibitor of mitochondrial complex I.30 Recently, preferential increase of nitration at a specific tyrosine residue of α-synuclein was observed in a cellular model of Parkinson disease.31 Significantly higher concentrations of nitrated and oxidized serum proteins were reported in smokers and lung cancer patients compared to those in healthy control subjects,32,33 supporting the role of oxidative and nitrosative stress induced

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xcessive production of nitric oxide (NO) is found in inflamed tissues by activated neutrophils and phagocytes. Reaction of NO with reactive oxygen species (ROS), produced during endogenous oxidative metabolism, generates the reactive nitrogen oxide species (RNOx), such as peroxynitrite, a biological nitrating agent of molecules, leading to 3-nitrotyrosine (3NT) formation on proteins.1,2 Both ROS and RNOx also cause various types of chemical modifications on other cellular biomolecules, including DNA and lipids.3,4 Peroxynitrite is not the only source of 3NT in vivo; nitryl chloride5 and peroxidases6−11 and certain metalloproteins10−14 may account for much of the 3NT formation at sites of inflammation. Acidification of nitrite might also contribute to tyrosine nitration in the stomach.15 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions. A number of inflammatory and neurodegenerative disorders have been associated with tyrosine nitration.16−19 Nitrated proteins are more susceptible to be degraded by the proteasome as a plausible mechanism of removing nitrated proteins in vivo.20 As a result, 3NT and its metabolites have been detected in human urine.21−23 Nevertheless, cellular tyrosine nitration is a low-frequency modification compared to protein phosphorylation. The frequency © 2012 American Chemical Society

Received: June 13, 2012 Accepted: August 29, 2012 Published: September 7, 2012 7881

dx.doi.org/10.1021/ac301597r | Anal. Chem. 2012, 84, 7881−7890

Analytical Chemistry

Article

by smoking and in carcinogenesis.34,35 In theses studies,32,33 however, levels of protein 3NT were quantified by Western blotting, which is not a very specific measurement. The recent advancement of mass spectrometry-based proteomics techniques has enabled investigation of the identity and sites of targeted nitroproteins.36 Because of the low abundance of tyrosine nitrated proteins, an antibody against 3NT is often used to enrich 3NT-containing proteins before Western blot or mass spectrometric analysis.37 Other enrichment strategies, including a series of reactions and derivatization, have been developed to tackle the low-abundant nitrated proteins in proteomic studies.36,38−40 Danielson and co-workers first employed the multiple reaction monitoring (MRM) method using a triple quadrupole mass spectrometer for relative quantification of 3NT-containing peptides in α-synuclein after immunoprecipitation.31 In this study, a total of 11 types and sites of post-translational modifications (PTMs) in hemoglobin induced by peroxynitrite were characterized by accurate mass measurement and multistage mass spectrometry. The extents of these modifications in hemoglobin freshly isolated from human blood were semiquantified by nanoLC−nanospray ionization linear ion trap mass spectrometry (nanoLC−NSI/MS) under the selected reaction monitoring (SRM) mode. The effect of cigarette smoking on the extents of these modifications was investigated.

Accurate Mass Analysis. The hHb solution treated with peroxynitrite (15 mM) was digested with trypsin, precipitated as described above, and reconstituted in 100 μL of 0.1% trifluoroacetic acid before analysis by a reversed phase nanoLC system connected to an LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, CA). 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 for fragmentation with a normalized collision energy value of 35% in the LTQ were selected. A repeat duration of 180 s was applied to exclude the same m/z ions from the reselection for fragmentation. Proteins were identified using the in-house MASCOT v2.3.02 search engine on the Swiss-Prot 56 human protein database. The mass tolerance was set to be 5 ppm for precursor and 0.8 Da for product ions. All MS/MS spectra were searched against the database for detecting variable modifications including the oxidation of methionine (+ 16), cysteine (+ 32 or + 48), nitrosylation (+ 29), and nitration (+ 45) of tyrosine or tryptophan residues, and one missed cleavage on trypsin was allowed. The cutoff score was set to 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. A volume of 4 μL of each sample was injected onto an LC system consisting of an UltiMate 3000 RSLCnano system (Dionex, Amsterdam, Netherlands) and a C18 precolumn (100 μm × 20 mm) packed in-house (Magic C18, 5 μm, 100 Å, Michrom BioResource, Auburn, CA), followed by separation using a C18 tip column (75 μm × 120 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 elution system started with 4% B for the first 2 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, maintained at 90% B for another 10 min at a flow rate of 300 nL/min. The conditions were equilibrated with 4% B for 20 min before the next run. 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. All MS2 experiments for peptide characterization were performed at a heated capillary temperature of 200 °C with a capillary voltage of 2.0 V, a source voltage of 1.5 kV, a tube lens voltage of 70 V, a source current of 100 μA, a normalized collision energy setting of 35%, and the ion gauge pressure of 6.4 × 10−6 Torr. Sequence Database Search and Data Analysis. To identify sites of modifications, hHb (0.1 mM) incubated with peroxynitrite (15 mM) was precipitated to remove excess reagent and digested with trypsin as described above. The digest was analyzed by nanoLC−NSI/MS/MS under the datadependent scan mode, automatically switched between full MS scan (m/z 350−2000) and MS/MS scan for the nine most abundant signals. Precursors detected twice within 30 s were put on a dynamic exclusion list (maximum of 50) for a period of 180 s. Ions with a charge state of 4 and higher were excluded. Peptide fingerprinting from the MS/MS data was performed with the aid of the TurboSEQUEST algorithm incorporated in BioWorks version 3.3 (Thermo Electron Corp., San Jose, CA) to correlate the data against the NCBI protein database (National Center for Biotechnology Information, Bethesda,

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EXPERIMENTAL SECTION Materials. Dithiothreitol (DTT), iodoacetic acid (IAA), and human hemoglobin (hHb) were purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin was from Promega Corporation (Madison, WI). All reagents are of reagent grade or above. Reaction of Human Hemoglobin with Peroxynitrite. A solution containing 0.1 mM of hHb from Sigma and 15 mM peroxynitrite in ammonium bicarbonate (25 mM) in a total volume of 0.2 mL was incubated at room temperature for 10 min under a nitrogen atmosphere. An aliquot (7.8 μL, equivalent to 50 μg of Hb) of the mixture was added to cold acetone (80 μL) and was allowed to stand at −20 °C for 15 min, followed by centrifugation at 0 °C for 20 min at 23 000g. The supernatant was removed, and the precipitate was airdried. Enzyme Digestion. The precipitated hemoglobin was dissolved in doubly distilled water and quantified by fluorescence described below. An equivalent to 50 μg of Hb was reconstituted in 80 μL of water, added 10 μL of ammonium bicarbonate (100 mM, final concentration, pH 8.0) and 10 μL of SDS (1.0%, final concentration), and incubated at 95 °C for 10 min. Cold acetone (900 μL) was added to the mixture, and it sat at −20 °C for 15 min and then centrifuged at 23 000g for 20 min. To the precipitate was added trypsin (10:1, w/w, 50 μL, 5 μg) and incubated at 37 °C for 18 h. Trifluoroacetic acid (0.1%, 50 μL) was added to the reaction mixture to stop the digestion. The trypsin digest was passed through a 0.22 μm Nylon syringe filter, and 4 μL of the solution was injected into the nanoLC−NSI/MS/MS system. In experiments with reduction/alkylation, a total volume of 100 μL of aqueous solution containing 50 μg of Hb, ammonium bicarbonate (100 mM, pH 8.0), SDS (1.0%), and DTT (10 mM) was incubated at 95 °C for 10 min, followed by alkylation with IAA (500 mM, 11 μL) with shaking at room temperature in the dark for 1 h. The solution was precipitated by cold acetone and digested with trypsin as described above. 7882



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centrifuged (800g, 10 min, 10 °C) to separate red blood cells from serum. Hemoglobin was isolated as reported.42 The isolated globin was dissolved in water and quantified by intrapolation to a calibration curve constructed from solutions containing various concentrations (0.02, 0.04, 0.06, 0.08, and 0.1 μg/μL) of standard hHb (100 μM) from Sigma Chemical Co. in hydrochloric acid (50 mM) and measured by the tryptophan-induced fluorescence at the excitation and emission wavelength at 280 and 353 nm, respectively.43,44 Study-Subjects. This study is approved by the Institutional Review Board of the National Chung Cheng University (IRB No. 100112902). The study-subjects were healthy individuals recruited from employees and students of the National Chung Cheng University, including 20 male smokers and 20 nonsmokers (11 male and 9 female). The mean (± standard deviation (SD)) age was 23.5 ± 5.9 for smokers and 25.0 ± 6.5 for nonsmokers. The mean (± SD) smoking index (number of cigarette per day × years smoked) of the study-subjects was 38.4 ± 67.9 in a range of 0−300. Statistical Analysis. GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, www. graphpad.com) was used for statistical analysis. The nonparametric Mann−Whitney test was used to analyze the extent of each modification between the 20 smokers and 20 nonsmokers. The correlation between the extent of each modification and the number of cigarettes smoked per day or smoking index was performed by the nonparametric Spearman correlation.

MD) with the following parameters: threshold, 1000; group scan tolerance, 1; minimum group count, 1; precursor charge state, auto; MSn level, auto. Monoisotopic mass was used for the search, and the mass tolerance for both peptides and fragment ions was set at 0.5 amu. Two miscleavage sites were allowed for digestion with trypsin at sites of arginine and lysine. All MS/MS spectra were searched against the database using the differential search parameters specified for detecting variable modifications including the oxidation of methionine (+ 16), cysteine (+ 32 or + 48), nitrosylation (+ 29), and nitration (+ 45) of tyrosine or tryptophan residues. The assignment of the peptide modifications was based on the following criteria: Peptides were accepted if they had a cross correlation (Xcorr) of at least 1.90, 2.2, and 3.75 for singly doubly, and triply charged ions,41 respectively, as well as the probability and sequence coverage scores accepted in the MS acquisition software. The ion peaks in the spectra were manually examined and assigned according to the charge state and derived mass. The MS3 experiments were performed by acquiring the daughter ion scan spectra of the most abundant b or y ion containing the modification in the MS/MS spectra with 35% normalized collision energy and at the parent ion at 2 m/z isolation width. Activation Q for collision-induced dissociation (CID) was set at 0.25 with an activation time of 30 ms. Semiquantification of Modified Peptides. The selected reaction monitoring (SRM) experiments were performed by selection of the precursor ion and acquisition of full-scan product ion spectra. The formation of a specific fragment ion from each precursor ion was used to construct the chromatogram. The specific SRM conditions for peptides containing tyrosine, methionine, cysteine, and their modifications are listed in Table 2S, 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 the corresponding reference (unmodified) peptide in the SRM chromatograms. Dose-Dependency of the Extents of PTM by Peroxynitrite or H2O2 Concentrations. Samples of commercial hHb (0.1 mM) incubated with various concentrations of peroxynitrite (0, 10, 20, 80, or 100 μM) in ammonium bicarbonate (25 mM) and potassium phosphate buffer (0.1 M, pH 7.4) at room temperature for 10 min were analyzed as described above. The experiments were performed in triplicates for each concentration. The dose-dependency was plotted as the peak area ratios versus the concentration of peroxynitrite. Samples of hHb (0.1 mM) isolated from a blood sample were treated with various concentrations of H2O2 (0, 10, 20, 100, or 200 μM) in potassium phosphate buffer (0.1 M, pH 7.4) at 37 °C for 1 h and analyzed as described above. The experiments were performed in triplicates for each concentration. The dose-dependency was plotted as the peak area ratios versus the concentration of H2O2. Stability of the PTMs. A 0.2 mL solution of peroxynitrite (0.1 mM)-treated hHb (0.1 mM) was dialyzed with a 1 K semipermeable membrane (Membrane Filtration Products, Inc. Seguin, TX) in 5 L of doubly distilled water at 4 °C overnight. The dialyzed Hb sat at 37 °C, and an equivalent of 50 μg of protein was removed after 0, 2, and 7 days for analysis by nanoLC−NSI/MS/MS after digestion. Isolation of Hemoglobin from Fresh Blood. The blood samples (10 μL) were collected fresh in a tube containing 10% (v/v) citrate-dextrose solution as an anticoagulant and

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RESULTS AND DISCUSSION The abundance of modified peptides in human blood is very low; thus, a hemoglobin sample was treated with peroxynitrite in vitro to obtain high extents of modifications for characterization purpose. The sample was digested with trypsin, and the peptides were analyzed by the sequence database search to identify the types and sites of modifications which were confirmed by accurate mass measurement. The PTMs induced by peroxynitrite were further characterized by fragment ions in the collision-induced dissociation (CID or MS2) and in the triple-stage (MS3) mass spectra. Subsequently, the selected reaction monitoring (SRM) transitions for the reference and modified peptides were optimized for nanoLC−NSI/MS/MS analysis. Finally, the extents of these PTMs in hemoglobin isolated from human blood samples were quantified relative to the reference peptides. Accurate Mass Analysis of Post-Translational Modifications in Peroxynitrite-Treated Hemoglobin. Characterization of post-translational modifications (PTMs) in peroxynitrite-treated human hemoglobin (hHb) was performed using the shotgun approach under the data-dependent scan mode. In this sample, hHb (0.1 mM) was reacted with peroxynitrite (15 mM) in the presence of physiological concentration of bicarbonate (25 mM). To circumvent false positive identifications of modified peptides, the mass tolerance of parent ions was set below 5 ppm and that for fragment ions was 0.8 Da for Mascot search. The difference between the experimental and calculated theoretical mass of these 11 modified peptides was between 0.0001 and 0.0097. The Mascot cutoff score was set at 20 and the scores of these modified peptides were all greater than 25. The MS/MS spectra of the modified peptides as well as their parent peptides were manually evaluated. The sequence coverage of this sample was 96% for α-globin and 89% for β-globin. As listed in Table 7883



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Table 1. Accurate Mass Analysis of Modified and Unmodified Peptides in Hemoglobin Treated with Peroxynitritea m/z exptl

z

M exptl

M calcd

ΔM

score

modification

17−31

VGAHAGE24YNOGAEALER

peptides from α-globin

779.8697

2

1557.7248

1557.7171

0.0077

27.73

17−31

VGAHAGE24YNO2GAEALER

787.8639

2

1573.7132

1573.7121

0.0011

77.47

− H + NO (Y, + 29) − H + NO2 (Y, + 45)

17−31 32−40 32−40 41−56

VGAHAGE24YGAEALER MOFLSFPTTK 32 MFLSFPTTK T42YNO2FPHFDLSHGSAQVK

765.3706 544.2793 536.281 939.9441

2 2 2 2

1528.7267 1086.5441 1070.5474 1877.8736

1528.7270 1086.5420 1070.5471 1877.8697

−0.0003 0.0021 0.0003 0.0039

80.82 40.73 50.45 42.19

41−56 62−90 62−90 100−127 100−127

917.4507 1004.8362 999.5024 1000.5417 1005.8714

2 3 3 3 3

1832.8869 3011.4868 2995.4854 2998.6032 3014.5923

1832.8846 3011.4771 2995.4821 2998.5950 3014.5899

0.0023 0.0097 0.0033 0.0082 0.0024

101.2 79.21 126.2 29.55 41.85

41−59 41−59 83−95 83−95 105−120 121−132

T42YFPHFDLSHGSAQVK VADALTNAVAHVDD76MOPNALSALSDLHAHK VADALTNAVAHVDD76MPNALSALSDLHAHK LLSH104CO2LLVTLAAHLPAEFTPAVHASLDK LLSH104CO3LLVTLAAHLPAEFTPAVHASLDK peptides from β-globin FFESFGDLSTPDAV55MOGNPK FFESFGDLSTPDAV55MGNPK GTFATLSELH93CO3DK GTFATLSELH93CDK LLGNVLV112CO3VLAHHFGK EFTPPVQAA130YNO2QK

1037.9765 1029.9773 735.3325 711.3382 884.4833 712.3480

2 2 2 2 2 2

2073.9385 2057.9400 1468.6505 1420.6619 1766.9521 1422.6815

2073.9354 2057.9405 1468.6504 1420.6657 1766.9502 1422.6779

0.0031 −0.0005 0.0001 −0.0038 0.0020 0.0036

78.06 106.7 56.38 45.06 59.88 50.74

121−132

EFTPPVQAA130YQK

689.8534

2

1377.6923

1377.6929

−0.0005

68.94

AA start-end

32

+ O (M, + 16) − H + NO2 (Y, + 45) + O (M, + 16) + O2 (C, + 32) + O3 (C, + 48) + O (M, + 16) + O3 (C, + 48) + O3 (C, + 48) − H + NO2 (Y, + 45)

a

A solution of human hemoglobin (0.1 mM) was incubated with peroxynitrite (15 mM) at room temperature for 10 min, followed by trypsin digestion and analysis by nanoLC−NSI/MS/MS as described in the Experimental Section.

As shown in Figure 1, several fragment ions with a mass shift of + 45 or + 28 (M − H + NO2 − NH3) or + 27 (M − H + NO2 − H2O) provide supportive evidence for nitration on the tyrosine residues (α-Tyr-24, α-Tyr-42, and β-Tyr-130). Table 1S in the Supporting Information lists the transitions for obtaining the MS3 spectra. Tyrosine nitrosylation at α-Tyr-24 was evidenced by a mass shift of + 29 of the molecular weight of the modified peptide relative to the parent peptide. In addition, the CID spectrum of t h e n it r o s o t y r o s in e- c o n t a in i n g p e p t id e V G A H A GE24YNOGAEALER at the [M + 2H]2+ ion at m/z 779.9 showed a mass difference of 192 between the singly charged b8′ (m/z 814.2) and b7 (m/z 622.2) as well as between y8′ (m/z 937.3) and y7 (m/z 745.3) fragment ions, indicating a nitrosotyrosine moiety (Figure 2A). Compared with the CID spectrum of the parent peptide (Figure 2B), the difference between b8′ and b8 and between y8′ and y8 are both + 29. In addition, the fragment ions containing nitrotyrosine in Figure 2A are 29 mass units greater than the corresponding fragment ions containing tyrosine in Figure 2B. Furthermore, the MS3 spectrum of the doubly charged b12′2+ ion at m/z 666.4 gave the [y12′ − NH3]2+ ion at m/z 657.8 and the y8′ − NH3 ion at m/z 920.8 (Figure 2C). The selected reaction monitoring (SRM) experiments were performed by selecting the precursor ion and acquiring full-scan product ion spectra. The elution of the modified peptides was analyzed by nanoLC−NSI/MS/MS with monitoring the fragmentation of the parent ion to the most abundant daughter ion in the SRM mode. The SRM transitions of the 11 PTMs were optimized and summarized in Table 2S, Supporting Information. The nanoLC−MS/MS chromatograms of nitrotyrosine-, nitrosotyrosine-, and tyrosine-containing peptides under the SRM mode showed that the modified peptides eluted later than their parent peptides by 2−3 min and that the nitrosylated peptide of α-Tyr-24 eluted slightly earlier than the

1, a total of 11 modifications were identified, including nitration on 3 tyrosine residues, nitrosylation on 1 tyrosine, oxidation of all 3 methionine residues as the sulfoxide, and oxidation of all 3 cysteine residues as the sulfonic acid plus 1 cysteine as the sulfinic acid. Identification of these PTMs followed the stringent criteria of Danielson et al.31 except that the immonium ion of 3NT at m/z 181 detected by the triple quadrupole mass spectrometer was not detected because it is in the low mass region limited by the ion trap mass spectrometer used in this study. Tyrosine Nitration and Nitrosylation. Among the six tyrosine residues in human Hb, nitration at α-Tyr-24, α-Tyr-42, and β-Tyr-130 was observed in the trypsin digest, along with nitrosylation at α-Tyr-24. In the trypsin digest, α-Tyr-140 and β-Tyr-145 residues were in the dipeptides located at the Ctermini of the α- and β-globin, respectively. Because these dipeptides were not retained in the LC system, we were unable to characterize them. The unmodified peptide containing Tyr35 (β-globin) as well as the corresponding nitrated peptide was one of the few peptides not detected in the trypsin digest. The nitrated peptides were characterized by a mass increase of 45 Da and a chromatographic delayed elution of ∼2−4 min compared to their parent peptides. Furthermore, an increase of 45 mass units was observed between the b or y ions of tyrosinecontaining fragments in the daughter ion spectra of these nitrated peptides compared to those of the parent peptides. Additionally, the mass difference of 208 mass units between the flanking b or y ions of tyrosine-containing fragments confirmed the presence of nitrotyrosine in the daughter ion spectra of these modified peptides. The CID spectra of these nitrotyrosine-containing peptides are consistent with those reported in H2O2/nitrite-treated hemoglobin.29 The MS3 spectra of the nitrotyrosine-containing peptides were obtained by collision of the selected fragment ions containing the modification, which are not necessarily the most abundant ions in the CID spectra. 7884



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Figure 2. MS2 scan spectra of (A) VGAHAGE24YNOGAEALER at m/z 779.90 and (B) VGAHAGE24YGAEALER at m/z 765.40. (C) MS3 spectrum of m/z 779.90 → 666.40 (y12′2+). The symbols “′”, “*”, and “o” indicate modification (nitrosylation), − NH3, and − H2O ions, respectively.

Figure 1. MS3 scan spectra of 3-nitrotyrosine-containing peptides: (A) VGAHAGE24YNO2GAEALER from m/z 787.80 to 674.40, (B) T42YNO2FPHFDLSHGSAQVK from m/z 940.00 to 817.50, and (C) EFTPPVQAA130YNO2QK from m/z 712.40 to 1046.30. The symbols “′”, “*”, and “o” indicate modification (nitration), − NH3, and − H2O ions, respectively.

nitrated peptide (Figure 3). The extent of modification was expressed by the ratio of peak area of the modified peptide over 7885



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methionine residues, Met-32, Met-76 in α-globin, and Met-55 in β-globin to the sulfoxide. The molecular weight of the modified peptides was highly accurate regarding to the theoretical value, with a 16 Da (+ O) increase compared to the respective parent peptides. The MS2 and MS3 spectra of the modified peptides were in agreement with the sulfoxide structure on the methionine residues (Figures 1S and 2S, Supporting Information). This finding is not surprising because methionine sulfoxide is a common modification found in proteins,45 and nature has evolved methionine sulfoxide reductase46 to circumvent its presence. Peroxynitrite is not only a nitrating agent but also an oxidizing agent, which has been shown to cause formation of methionine sulfoxide.47 As analyzed by nanoLC−NSI/MS/MS, methionine sulfoxidecontaining peptides eluted 2−4 min earlier than the parent peptides (Figure 3S, Supporting Information). Cysteine Oxidation. In peroxynitrite-treated hemoglobin, cysteine oxidation products were characterized as sulfonic acid on α-Cys-104, β-Cys-93, and β-Cys-112, together with sulfinic acid on α-Cys-104. Both sulfonic and sulfinic acids are redoxsensitive cysteine modifications,48 and they have been identified in cellular proteins.49,50 The sulfinic acid was previously thought to be an irreversible modification, and the biological reduction systems of the sulfinic acid have been identified.51,52 The peptides containing sulfonic and sulfinic acid showed a mass shift of + 48 (+ O3) and + 32 (+ O2) Da, respectively, compared to the parent peptides (Table 1), as well as the b and y fragment ions containing cysteine in the MS2 and MS3 spectra

Figure 3. nanoLC−MS/MS chromatograms of tyrosine- and 3nitro(so)tyrosine-containing peptides under the selected reaction monitoring mode.

the sum of peak areas corresponding to the modified and the parent peptide in the LC−MS/MS chromatograms. Methionine Oxidation. Also characterized in this peroxynitrite-treated hemoglobin sample was oxidation of all three

Table 2. Extent of Modifications in Hemoglobin Processed with and without Reduction/Alkylation no DTT/IAAa extent of modificationsc,d (%) 24 NO

VGAHAGE Y GAEALER VGAHAGE24YNO2GAEALER VGAHAGEYGAEALER T42YNOFPHFDLSHGSAQVK T42YNO2FPHFDLSHGSAQVK TYFPHFDLSHGSAQVK EFTPPVQAA130YNO2QK EFTPPVQAAYQK 32 O M FLSFPTTK MFLSFPTTK VADALTNAVAHVDD76MOPNALSALSDLHAHK VADALTNAVAHVDDMPNALSALSDLHAHK FFESFGDLSTPDAV55MOGNPK FFESFGDLSTPDAVMGNPK LLSH104CO3LLVTLAAHLPAEFTPAVHASLDK LLSH104CO2LLVTLAAHLPAEFTPAVHASLDK TYFPHFDLSHGSAQVK LLSH104C*LLVTLAAHLPAEFTPAVHASLDK GTFATLSELH93CO3DK GTFATLSELH93CO2DK VNVDEVGGEALGR GTFATLSELH93C*DK LLGNVLV112CO3VLAHHFGK LLGNVLV112CO2VLAHHFGK VLGAFSDGLAHLDNLK LLGNVLV112C*VLAHHFGK

with DTT/IAAb extent of modificationsc,d,e (%)

ratiof (%)

0.00047 0.023

0.00020 0.018

42 76

0.037 0.040

0.037 0.029

0.0030

0.00035

4.69

4.68

100

4.01

3.98

99

5.98

5.79

97

1.3 0.83

0.81 (12) 0.27 (3.9)

62 32

6.7 0.26

4.8 (9.5) 0.18 (0.36)

72 67

1.2 0.016

0.71 (15) 0.0032 (0.0064)

58 20

100 73 12

a The experiments were performed with the commercial hemoglobin following procedures described in the Experimental Section. bThe experiments were performed with DTT and IAA as described in the Experimental Section. cMean of triplicate experiments. dThe native peptides shown in bold were used as reference peptides. eQuantification using the alkylated peptides (shown in italic) as reference peptides is shown in the parentheses. fThe ratio was calculated by dividing the extent of modification performed with DTT/IAA by the extent of modification performed without DTT/IAA and shown as a percentage.

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procedure is not suitable for quantitative studies involving endogenous tyrosine nitration/nitrosylation and cysteine oxidation. Among the PTMs identified and quantified in human blood, only one tyrosine-containing peptide is nitrosylated with three nitrated peptides and only one sulfinic acid is identified with three sulfonic acid-containing peptides. As mentioned above, the identification of these PTMs followed stringent criteria including good mass accuracy, similar fragmentation pattern of CID spectra to the parent peptide, flanking b- or y-fragment ions containing the modification, and chromatographic shift on LC−MS/MS. The possible presence of nitrosylation of α-Tyr42 and β-Tyr-130 and sulfinic acid formation on β-Cys-93 and β-Cys-112 was carefully examined. In the data-dependent scan of peroxynitrite-treated hemoglobin, the nitrosylated peptide of β-Tyr-130 was not matched by Bioworks, which was not surprising because this peptide is the least preferred site of nitration, and the extent of nitrosylation is much lower than that of nitration as in α-Tyr-24. The probability values for sulfinic acid-containing peptides of β-Cys-93 and β-Cys-112 were too high, and their CID spectra did not follow the same pattern as those of the corresponding nitrated and parent peptides, which disqualified them as sites of valid PTM. A daughter ion scan experiment was performed on the peroxynitrite-treated hemoglobin to find the daughter ion for the SRM transitions. As shown in the nanoLC−NSI/MS/MS chromatograms (Figure 8SA, Supporting Information), only the nitrosylated α-Tyr-24 eluted ∼1 min earlier than the nitrated peptide and later than the parent peptide. Both “simulated” nitrosylated α-Tyr-42 and β-Tyr-130 eluted very close to their nitrated peptides (Figure 8SB, Supporting Information). Moreover, in the experiment including DTT/ IAA procedure, the extent of “nitrosylated” α-Tyr-42 was nearly unchanged, while those of nitrosylated α-Tyr-24 and the three nitrated peptides decreased from 24% to 88% (Table 2). In addition, “nitrosylated” α-Tyr-42 was disqualified by failing to elute earlier than the nitrated peptide (Figure 8SA, Supporting Information). These results are summarized in Table 3.

of the modified peptides (Figures 4S and 5S, Supporting Information). The sulfinic acid-containing peptide eluted (44.60 min) ∼1 min earlier than the one with sulfonic acid (45.46 min), as shown in Figure 6S, Supporting Information. Relative Quantification of Post-Translational Modifications in Peroxynitrite-Treated Hemoglobin. Although the ionization and fragmentation efficiencies might be different for the modified and unmodified peptides, the extent of modification can be estimated by semiquantitative measurement. Using the optimal SRM conditions, the extent of PTM was 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. The reference peptides are the parent peptides for tyrosine-and methionine-containing peptides. The signals for the cysteine-containing parent peptides were very small without reduction followed by alkylation of the cysteine residues. Normally, reduction was performed by dithioltreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) before alkylation of the cysteine residues by agents such as iodoacetic acid (IAA). However, we and others reported that 3NT could be partially reduced to 3-amiotyrosine by DTT under the conditions of the standard DTT/IAA procedure (at 95 °C) and at 37 °C.28,29,53 Because the three cysteine residues in native hemoglobin exist as free thiols, the reduction/alkylation step was omitted. Although hemoglobin was denatured with SDS only, the sequence coverage after digestion was very high, i.e., 96% and 89% for α- and β-globin, respectively. However, the signals corresponding to the three cysteine-containing parent peptides were very small, possibly due to spontaneous oxidation and/or formation of disulfides with themselves or with each other during protein digestion. Alternatively, native peptides in the trypsin digest of hemoglobin eluted closely to the modified peptides were chosen and used as reference peptides for quantification of the extent of sulfonic and sulfinic acid-containing peptides. Under the SRM mode, elution of the modified peptides and their reference peptides were shown in Figure 6S, Supporting Information. The concept of the “native reference peptide” method has been applied to phosphopeptides54 as well as nitropeptides.39 The native reference peptides with similar polarity, and possibly similar ionization efficiency, to the modified peptide correct for variations in protein amounts and peptide recovery in the digest preparation procedure. Relative quantification using native reference peptides allows comparison between samples but it does not provide the absolute extent of modification. As analyzed by nanoLC−NSI/MS/MS under the SRM mode, the 11 PTMs identified in peroxynitrite-treated hHb were semiquantified. The extents of tyrosine nitration, but not nitrosylation, were found to increase dose-dependently on peroxynitrite concentration (Figure 7S, Supporting Information), but methionine and cysteine oxidation were not (data not shown). To examine the effect of reduction/alkylation steps on the extents of tyrosine nitration/nitrosylation, methionine oxidation, and cysteine oxidation, the experimental procedures were performed on commercial human Hb with and without DTT/ IAA. Table 2 showed that in the presence of DTT, the extents of nitration on α-Tyr-24 and α-Tyr-42 decreased by ∼25%, while that for β-Tyr-130 decreased by 88%. Methionine oxidation was not affected by DTT. Sulfonic acid formation decreased by an average of 36%, while sulfinic acid on α-Cys104 decreased by 68%. These results imply that the DTT/IAA

Table 3. Criteria for Nitrosylation of α-Tyr-42 and β-Tyr130 and Sulfinic Acid Formation on β-Cys-93 and β-Cys112a data-dependent scan CID pattern fragment ions chromatographic shift decrease with DTT/IAA a

α-42TyrNO

β-130TyrNO

β-93CysO2

β-112CysO2

√ √ × × ×

× × × − −

× × √ × √

× × √ × √

√, pass; ×, failed; −, not analyzed.

Extent of Oxidative Modifications in H2O2-Treated Hemoglobin. In order to compare the extent of oxidative modifications by peroxynitrite with that derived from other ROS, hHb was incubated with various concentrations of H2O2. Figure 9SA in the Supporting Information showed that methionine oxidation was merely affected by H2O2 up to 200 μM. On the other hand, cysteine oxidation increased dosedependently with H2O2 (Figure 9SB, Supporting Information). The reason might be that the extents of methionine oxidation were already too high to be affected by additional H2O2. Digestion of Hemoglobin under Aerobic and Anaerobic Conditions. The dissolved oxygen in the aqueous 7887



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Figure 4. The extent of (A) α-Tyr-24 and (B) α-Tyr-42 nitration in smokers and in nonsmokers.

solution might contribute to the extent of peroxynitrite-induced PTMs formation in hHb during enzyme digestion (37 °C, 18 h). Thus, enzyme digestion under anaerobic conditions was performed. Doubly distilled water was boiled for 15 min, cooled to room temperature, and bubbled argon for 5 min before its use for making solutions. Comparing the extents of PTMs formation with those under aerobic conditions without deoxygenation of water, no significant difference in the extents of modification on these PTMs (data not shown) was observed. Therefore, regular doubly distilled water was used throughout this study. Stability of PTMs. The possibility that nitrosotyrosine and sulfinic acid be oxidized to the respective nitrotyrosine and sulfonic acid during digestion was investigated. The stability of these 11 PTMs was examined on peroxynitrite-treated hHb, which was dialyzed overnight prior to incubation at 37 °C for up to 7 days, followed by trypsin digestion and nanoLC−NSI/ MS/MS analysis. The result showed no significant change in the extents of modification on these PTMs (data not shown), suggesting that they are stable modifications. Post-Translational Modifications in Hemoglobin Isolated from Fresh Blood. A total of 40 human blood samples, including 20 smokers and 20 nonsmokers, were analyzed for these 11 PTMs by the nanoLC−NSI/MS/MS using the SRM method. Only 10 μL of blood was processed for Hb isolation. After quantification of Hb concentration, 50 μg of Hb was digested and 1/25 of the solution was subjected to nanoLC− NSI/MS/MS analysis. That is, only an equivalent of 0.01−0.02 μL of blood was analyzed. Because of the involvement of the precipitation step during isolation of Hb, it is difficult to obtain precipitated Hb starting from a volume of blood less than 10 μL. The results showed that the extents of nitration on α-Tyr-24 and α-Tyr-42 were significantly higher in smokers than in nonsmokers using the nonparametric Mann−Whitney test with p values of 0.0061 and 0.0040, respectively (Figure 4 and Table 3S, Supporting Information). Moreover, the extent of nitration on α-Tyr-24 and α-Tyr-42 correlates significantly with the number of cigarettes smoked per day with a Spearman correlation coefficient (r) of 0.3832 (p = 0.0147) and 0.4367 (p = 0.0048), respectively. The smoking index, defined as the number of cigarettes smoked per day times years smoked, is also associated with the extent of nitration on α-Tyr-24 and αTyr-42 with a Spearman r of 0.4367 (p = 0.0076) and 0.3730 (p = 0.0178), respectively (Table 4). Furthermore, the extent of nitration on α-Tyr-24 and α-Tyr-42 correlated with each other

Table 4. Statistical Correlation of the Extent of Nitration on α-Tyr-24 And α-Tyr-42 with the Number of Cigarettes Per Day and Smoking Indexa 24 NO2

Y

Spearman rb Spearman rb a

42 NO2

Y

Number of Cigarettes Per Day 0.3823 (0.0147) 0.4367 (0.0048) Smoking Index 0.4157 (0.0076) 0.3730 (0.0178)

n = 40. bThe two-tailed p values are shown in the parentheses.

with a Spearman r of 0.6161 (p < 0.0001) (Figure 10S, Supporting Information). These results suggest that the extent of nitration on α-Tyr-24 and α-Tyr-42 might be used to assess the nitrative damage to human blood hemoglobin by cigarette smoke. The extents of nitration on α-Tyr-24, α-Tyr-42, and β-Tyr130 in these 40 samples were 0.0298% ± 0.0366%, 0.0028% ± 0.0019%, and 0.0284% ± 0.0497% (mean ± SD), respectively, which gives an average of 0.020% nitration (Table 3S, Supporting Information). This value is 8 times higher than the reported average 3NT content of 24 μmol/mol Tyr in hemoglobin of 12 normal healthy human subjects, in which hemoglobin was hydrolyzed under exhaustive enzyme digestion conditions to release free amino acids 3NT and Tyr from protein, followed by LC−MS/MS analysis.55 The discrepancy might be due to the following factors: First, the extents of nitration on the other three tyrosine residues, i.e., α-Tyr-140, βTyr-35, and β-Tyr-145, were not determined. The biological nitration is a selective process.16,19 Only certain proteins and certain tyrosine residues are preferentially nitrated. It is evidenced in this study that the extents of nitration of α-Tyr42 are approximately 10 times lower than those of α-Tyr-24 and β-Tyr-130. Second, the extent of nitration determined by semiquantification is not equal to the molar content of 3NT unless the molar response of the peaks of 3NT- and the corresponding Tyr-containing peptides are the same. In the future, absolute quantification of PTMs by using synthetic peptides of interest will be the solution. At present, semiquantitaive measurement provides a screening of which modification on which residue should be investigated further. Historically, hemoglobin adducts has been used to monitor exposure of mutagens and carcinogens for cancer risk assessment studies.56−58 These chemicals and/or their reactive metabolites modify the N-terminal amine groups of the Hb subunits. Quantification of these protein adducts are achieved 7888



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by Edman degradation of the adducted N-terminal valine residues, followed by GC−MS (after derivatization) or LC−MS analysis.56−58 In addition, hemoglobin adducts are relatively stable as there are no active repair systems in blood for their removal. Hemoglobin is a rich source of protein from blood as 1 mL of blood contains ∼150 mg of Hb in red blood cells and 30 mg of serum albumin. The half-life of human Hb is 63 days, longer than that of serum albumin (20 days).59,60 The relatively long life span of human Hb (∼4 months) and the accessibility in large amounts make it suitable as an exposure monitor. In addition, the 3-NT content of hemoglobin in healthy human subjects is about 10-fold higher than that of albumin in plasma.55 This highly sensitive and specific assay only requires hemoglobin isolated from ∼10 μL of blood. Without the use of immunoaffinity enrichment, a total of 11 PTMs were identified and semiquantified. Thus, measurement of these PTMs in hemoglobin might be used as biomarkers for evaluating oxidative and nitrative stress in vivo and their potential in disease development and prevention.

CONCLUSIONS The reactive nitrogen oxide species-induced post-translational modifications were characterized in peroxynitrite-treated hemoglobin, which include nitration and nitrosylation of tyrosine, sulfoxide formation on methionine, and sulfonic acid and sulfinic acid formation on cysteine residues. These modifications were confirmed at the peptide level by the accurate mass measurement as well as the MS2 and MS3 mass spectra, and they were semiquantified in hemoglobin freshly isolated from human blood. The extents of tyrosine nitration on α-Tyr-24 and α-Tyr-42 positively correlated with cigarette smoking and with the smoking index. To our knowledge, this is the first report of correlation of cigarette smoking with 3NT at a specific site in hemoglobin. Only one drop of blood (∼10 μL) is needed for this nanoLC−NSI/MS/MS analysis, which may provide feasible biomarkers for smoking-induced nitrative stress in clinical studies. ASSOCIATED CONTENT

S Supporting Information *

(1) MS2 and MS3 spectra of methionine- and cysteinecontaining peptides and (2) the extent of modification in hemoglobin isolated from human blood. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (886) 5-242-8176. Fax: (886) 5-272-1040. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Mr. Ming-Feng Chen of the Instrument Development Center of National Cheng Kung University for performing accurate mass measurement. This work was supported by National Science Council of Taiwan (Grants NSC 97-2113-M-194-007-MY3 and NSC 100-2113-M-194002-MY3) and National Chung Cheng University (to H.J.C.C.). 7889



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Reactive Nitrogen Oxide Species-Induced Post-Translational

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