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Analysis of chlorination, nitration, nitrosylation of tyrosine, and oxidation of methionine and cysteine in hemoglobin from type 2 diabetes mellitus patients by nanoflow liquid chromatography tandem mass spectrometry Hauh-Jyun Candy Chen, Ya-Fen Yang, Pang-Yen Lai, and Pin-Fan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02663 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Analytical Chemistry

Analysis of chlorination, nitration, nitrosylation of tyrosine and oxidation of methione and cysteine in hemoglobin from type 2 diabetes mellitus patients by nanoflow liquid chromatography tandem mass spectrometry

Hauh-Jyun Candy Chen,*1 Ya- Fen Yang1, Pang-Yen Lai1, and Pin-Fan Chen2

1

Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan 2

Division of Metabolism and Endocrinology, Department of Internal Medicine, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Chia-Yi 62247, Taiwan

*To whom correspondence should be addressed. Phone: 886-5-272-9176. Fax: 886-5-272-1040. E-mail: [email protected].

Keywords: 3-chlorotyrosine, diabetes mellitus; hemoglobin; LC-MS/MS; post-translational modification; Abbreviations: DTT, dithioltreitol; HOCl, hypochlorous acid; IAA, iodoacetic acid; nanoLCNSI/MS/MS, nanoflow liquid chromatographynanospray ionization tandem mass spectrometry; PTM, post-translational modification; REOM, relative extents of modification; SRM, selected reaction monitoring; T2DM, type 2 diabetes mellitus. Novel aspect: simultaneous analysis of multiple post-translational modifications in hemoglobin from type 2 diabetic patients 1

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Abstract The post-translational modification of proteins by endogenous reactive chlorine, nitrogen and oxygen species is implicated in certain pathological conditions, including diabetes mellitus. Evidence showed that the extents of modifications on a number of proteins are elevated in diabetic patients. Measuring modification on hemoglobin has been used to monitor the extent of exposure. This study develops an assay for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin and to examine whether the level of any of these modifications are higher in poorly controlled type 2 diabetic mellitus patients. This mass spectrometry-based assay used the bottom-up proteomic strategy. Due to the low amount of endogenous modification, we first characterized the sites of chlorination at tyrosine in hypochlorous acid-treated hemoglobin by an accurate mass spectrometer. The extents of chlorination, nitration, and oxidation of a total of twelve sites and types of modifications in hemoglobin were measured by nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry under the selected reaction monitoring mode. Relative quantification of these PTMs in hemoglobin extracted from blood samples shows that the extents of chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in diabetic patients (n=19) than in nondiabetic individuals (n=18). After excluding the factor of smoking, chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in the nonsmoking diabetic patients (n=12) than in normal nonsmoking subjects (n=11). Multiple regression analysis performed on the combined effect of age, BMI, and HbA1c showed that the diabetes factor HbA1c contributes significantly to the extent of chlorination on α-Tyr-24 in nonsmokers. In addition, age contributes to oxidation

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at -Met-32 significantly in all subjects and in nonsmokers. These results suggest the potential of using chlorination at α-Tyr-24-containing peptide to evaluate protein damage in nonsmoking type 2 diabetes mellitus.

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Introduction Humans are constantly exposed to reactive species produced endogenously via metabolism or by the innate immune systems. Among them, reactive halogen species, including hypochlorous acid (HOCl) is a chlorinating oxidant that possesses potent antibacterial, antiviral, and antifungal properties and they play key roles in the host defense system against microorganisms and during chronic infection and inflammation.1 Hypochlorous acid is produced via oxidation of chloride anion (Cl), existing in high concentration in the plasma (100 mM), by hydrogen peroxide (H2O2) catalyzed by myeloperoxidase.2,3 Modification of protein by HOCl forms 3-chlorotyrosine,4 which is considered as a specific biomarker of HOCl-induced protein modification.5 Excess HOCl is produced in inflamed tissues. After mildly allergic asthmatic patients were challenged with allergens, 3-chlorotyrosine content in bronchoalveolar lavage proteins increased 2-3 folds.6 Myeloperoxidase is abundant in leukocytes, which also catalyzes oxidation of nitrite anion by H2O2 to produce nitrating agents such as peroxynitrite.7,8 At the site of inflammation, these potent oxidants react with biomolecules such as proteins and DNA, leading to the accumulation of cellular damage that is implicated in many human pathological conditions. Nitration of DNA leads to formation of 8-nitropurines, which undergo spontaneously depurination giving rise to apurinic site, a mutagenic lesion.9-11 Nitration of protein forms 3-nitrotyrosine which is recognized as a biomarker for nitrative and oxidative stress associated with diseases including smoking, inflammatory diseases, neurodegenerative disorders, and cancers.12-16 Diabetes mellitus (DM) is a disease associated with oxidative stress and inflammation.17 Evidences revealed a relationship between oxidative stress and the pathogenesis of diabetic

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complications.17,18 Reactive nitrogen species, such as peroxynitrite and H2O2/nitrite, are oxidants causing nitration along with oxidation on methionine and cysteine residues in human hemoglobin.16,19 Levels of 3-nitrotyrosine were higher in plasma and urine of diabetic patients.18,20,21 Moreover, the extent of nitration on apolipoprotein AI is increased in diabetic patients with coronary artery disease.22

The contents of nitrated and chlorinated apolipoprotein

A-I in the serum of diabetic subjects were also increased.23,24 Recent advances in biological mass spectrometry empower studies of protein post-translational modifications (PTMs), which are present in low abundance, in both qualitative and quantitative aspects.25,26

Because oxidative stress is involved in the development of various

diseases, redox proteomics strategies enable discovery of protein modifications as biomarker in disease progress allowing early detection and diagnosis in various disease states.27,28 In this study, we developed an assay based on nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry (nanoLC /MS/MS) under the selected reaction monitoring (SRM) mode for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin to examine whether the level of these PTMs in poorly controlled type 2 diabetic patients (T2DM) is associated with the disease state.

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Materials and Methods

Materials. Dithiothreitol (DTT) and human hemoglobin (hHb) were purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin was from Promega Corporation (Madison, WI). All reagents used were of reagent grade or above. Isotopically labeled peptide MFL(13C6)SFPTTK was synthesized by Kelowna International Scientific Inc. (Taipei, Taiwan). Incubation of Human Hemoglobin with Hypochlorous Acid. A solution containing commercial hHb (0.1 mM) and sodium hypochlorite (100 M) in potassium phosphate buffer (100 mM, pH 7.4) in a total volume of 0.2 mL was incubated at 37 C for 20 min, followed by quenching with ammonium bisulfite (10 times molar excess of hypochlorite). Ten times volume of cold acetone was added to precipitate hemoglobin and the mixture was kept at 20 C for 15 min, followed by centrifugation at 23,000g at 0 C for 20 min to remove excess hypochlorous acid. Isolation and Quantification of Globin from Blood. Blood was freshly collected and contained in a tube containing citrate-dextrose solution (10%, v/v) as the anticoagulant, and it was centrifuged at 800g for 10 min at 10 C to isolate red blood cells. Globin isolated was quantified by interpolation into a calibration curve constructed from solutions of standard hHb in HCl (50 mM) measuring fluorescence excited at 280 nm and emitted at 353 nm.16,29 Trypsin Digestion of Globin. Fifty micrograms of isolated globin was dissolved in 100 L of ammonium bicarbonate (100 mM, pH 8.0) in the presence of 1.0% SDS. After the solution was incubated at 95 C for 10 min, it was added 900 L of cold acetone and let it sit at 20 C for 15 min. After centrifugation at 23,000g at 0 C for 20 min, the supernatant was discarded. To the precipitate was added trypsin (5 g) in 50 L of ammonium bicarbonate (100 mM, pH 8.0) 7



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and incubated at 37 °C for 18 h. The reaction was quenched with trifluoroacetic acid (0.1%, 50 L) and the solution was passed through a 0.22 m Nylon syringe filter, and 4 L of the solution was analyzed by the nanoLC /MS/MS system described below. In experiments with iodoacetic acid, after globin was heating with SDS, eleven microliters of 0.5 M iodoacetic acid was added to the solution with shaking at room temperature in the dark for 1 h, followed by precipitation with cold acetone and trypsin digestion. Characterization of Hemoglobin Chlorination. The hemoglobin solution incubated with hypochlorous acid (100 M) was digested with trypsin as described above. An aliquot equivalent of 0.5 g of globin digest was analyzed by a 75 μm × 250 mm Thermo Scientific Acclaim PepMap100 C18 Nano LC column eluting with a linear gradient from 8% mobile phases to 35% mobile phase B in 40 min with a flow rate of 300 nL/min. The mobile phases A and B were composed of 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile, respectively. The column was connected to an LTQ Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany). The peptides were analyzed using the positive ion mode with nanospray ionization interface with a spray voltage of 1.6 kV. The mass spectrometer was operated under a data-dependent scan mode using a rate of 30 ms/scan, in which one full scan is with m/z 350−1600 in the Orbitrap at a resolution of 60,000 at m/z 400. The five most intense peaks for fragmentation in the LTQ were selected with a normalized collision energy value of 35%. To exclude the same m/z ions from the reselection for fragmentation, a repeat duration of 90 s was applied. Post-translational modifications of hemoglobin were identified using the MASCOT Daemon 2.5.1

server

search

engine

on

the

Swiss-Prot

56

human

protein

database

(uniprot_sprot_2015_1.fasta (547,357 sequences) released Dec., 2014). The mass tolerance was

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set to be 10 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 (+ 32or + 48), chlorination (+ 34), and dichlorination (+68) 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. Relative Quantification by NanoLC-NSI/MS/MS. Two micrograms of each sample was injected onto an LC system consisting of an UltiMate 3000 RSLCnano system and a C18 precolumn (100 μm × 20 mm) packed in-house (Magic C18), followed by separation using a C18 tip column (75 μm × 120 mm) packed in-house (Magic C18AQ, 5 μm). 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 3 min, followed by a linear gradient from 4% B to 40% B in the next 37 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 column was coupled to an LTQ linear ion trap mass spectrometer equipped with the nanospray ionization (NSI) interface. The mass spectrometry conditions were as reported previously.16 The selected reaction monitoring transitions listed in Table S1 were used for relative quantification of the extent of modifications. The experiment for each sample was performed in triplicates. Dose-Dependent Formation of Chlorinated Peptides. A solution containing commercial hHb (0.1 mM) and sodium hypochlorite (0, 50, 100, or 1000 M) in potassium phosphate buffer (100 mM, pH 7.4) in a total volume of 0.2 mL was incubated at 37 C for 20 min, followed by quenching with ammonium bisulfite. After precipitation by cold acetone to remove excess

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hypochlorous acid, it was digested with trypsin as described above. The experiments were performed in triplicates for each concentration of hypochlorous acid. The dose-dependent formation of chlorinated peptides was plotted as the peak area ratios versus the concentration of hypochlorous acid. Extent of Artifactual Methionine Oxidation. Globin extracted from three blood samples was added SDS, heated, and precipitated as described above. To the precipitate was added MFL(13C6)SFPTTK (31 pmol) and digested with trypsine as described above. The extent of modification was analyzed by nanoLC-NSI/MS/MS as described above, with the addition of MFL(13C6)SFPTTK using m/z 539.44 (+2)  278.99 (+1) and the corresponding methionine sulfoxide using 547.44 (+2)  295.15 (+1) transitions. Study-Subjects. This study was performed with the approval from the Institutional Review Boards (IRB) of Buddhist Dalin Tzu Chi General Hospital (IRB No. B10203014-1) to recruit 19 poorly controlled type 2 diabetic mellitus (T2DM) patients (including 7 smoking males, 4 nonsmoking males, and 8 nonsmoking females). The average age was 54.5  15.2 ( SD) years, and their HbA1c levels averaged 9.5%  1.3% ( SD) ranging between 7.0% and 12.1%. Their body-mass index (BMI) averaged 28.1  4.7, ranging from 18.4 to 28.3. Recruiting nondiabetic subjects were approved by the IRB of the National Chung Cheng University (IRB No. 100112902). The nondiabetic subjects included 14 male (7 smokers) and 4 nonsmoking female workers and students of NCCU with the mean age being 22.2  1.4 ( SD) years. Their body-mass index (BMI) averaged 21.6  2.7, ranging from 18.4 to 28.3. Statistical Analysis. Statistical analysis was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software (San Diego, CA, www.graphpad.com). The comparison of

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the extents of each modification in the T2DM and the control group was performed by nonparametric MannWhitney test. The correlation between the extent of each modification and the HbA1c percentage or age or BMI was performed by the nonparametric Spearman correlation. Multiple regression analysis was performed to evaluate the contribution of HbA1c or age or BMI to the extent of modification.

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Results and Discussion Identification of Chlorinated Peptides. Due to the low abundance of endogenous modifications on hemoglobin, the sites and types of chlorination were identified in hemoglobin incubated with HOCl in vitro. After the HOCl-treated hemoglobin was digested with trypsin, the peptide mixture was analyzed by nanoflow LC connected to a high resolution LTQ-Orbitrap mass spectrometer under the data-dependent scan mode. In a solution of human hemoglobin (0.1 mM) incubated with HOCl (0.1 mM) at 37 C for 20 min at pH 7.4, chlorination at -Tyr-24 and -Tyr-130 were identified

(Table 1). Because

HOCl is an oxidizing agent, oxidative modifications, including as the methionine sulfoxide at the three methionine residues and as the sulfonic and sulfinic acids of three cysteine residues, were also identified in peroxynitrite-treated hemoglobin.16,19 The mass differences between the theoretical and experimental molecular weight (M) were all less than 4 ppm with the Mascot score of greater than 20. All the collision-induced dissociation (CID) spectra of the modified peptides were manually examined and compared to the corresponding unmodified peptides to ensure correct characterization. Chlorination at -Tyr-24-containing peptide VGAHGE24YClGAEALER was characterized by the CID spectrum of the doubly charged molecular ion at m/z 782.48 (Figure 1a) and compared with that of the unmodified parent ion at m/z 765.40 (Figure 1b). The mass difference between the y8+1 ion at m/z 942.38 and the y7+1 ion at m/z 745.43 is 196.95, accounting for a chlorotyrosine moiety. In addition, the mass difference between the y8+1 and y8+1 ions and between the b8+1 and b8+1 ions (Figure 1b) is 33.97, indicating the substitution of a hydrogen atom by a chlorine atom. The total ion mass spectrum of this peptide showed the [M+2H]+2 ion

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at m/z 782.24 and that with chlorine-37 isotope ([M*+2H]+2) at m/z 783.11 with an intensity ratio of ca. 3:1 (Figure S1, Supporting Information), providing supporting evidence for the presence of a chlorine atom in this peptide. Relative Quantification of Hemoglobin Modifications. The relative extents of chlorination, nitration, and nitrosylation of tyrosine, as well as oxidation of cysteine and methionine, were simultaneously measured in the tryptic digest of globin by nanoLC-NSI/MS/MS under the SRM mode. Native peptides intrinsically present in the globin trypsin digest which eluted closely to the modified peptides were used as the “native reference peptide (NRP)”.30 Using the peptide present in the protein digest (native peptide) as the reference peptide allows corrections for variations in the amount of protein used and in the peptide recovery during the digestion procedures. The reference peptides should not contain a miscleavage and it should be consistently present in the digest. For tyrosine- and methionine-containing peptides, the reference peptides are the unmodified parent peptides. The fragment patterns of the modified peptide are similar to those of the unmodified parent peptide. The fragment ions chosen for the modified peptides all include modifications and they are consistent with those for the parent and unmodified ions. The SRM transitions and the retention times for the modified and unmodified peptides are listed in Table S1, Supporting Information. The relative extents of modifications (REOM) are 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. For instance, there are three modifications identified on -Tyr-24-containing peptide. The relative extent of chlorination (REOMCl) on this peptide is expresses as the peak area of chlorinated peptide (ACl) divided by the sum of the peak areas of unmodified tyrosine-containing

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parent peptide (AY), chlorinated, nitrosylated (ANO), and nitrated (ANO2) peptides (Equation 1). For tyrosine nitrosylation, nitration, and methionine sulfoxide formation, the relative extent of modifications are expressed in Equations 24. REOMCl = ACl / (AY + ACl + ANO + ANO2)

(Equation 1)

REOMNO = ANO / (AY + ACl + ANO + ANO2)

(Equation 2)

REOMNO2 = ANO2 / (AY + ACl + ANO + ANO2)

(Equation 3)

REOMsulfoxide = Asulfoxide / (AM + Asulfoxide)

(Equation 4)

We examined the possibility of using the alkylated cysteine-containing peptides as the reference peptide for measuring the REOM on the cysteine residues. However, the modifications analyzed in this study are associated with oxidative stress; the commonly used reduction/alkylation procedures with dithioltreitol (DTT) and iodoacetic acid (IAA) led to decrease in their REOMs to various extents, as we reported previously.16 Because there are no disulfides in native hemoglobin, we used the alkylating agent IAA alone without the reducing agent. The results show that addition of IAA affects these REOMs to different extents (Table S2, Supporting Information), probably due to overalkylation to the amino group(s) of the peptide as evidenced by the reported MS/MS spectra.31 Therefore, to quantify modifications on cysteine-containing peptides, native peptides intrinsically present in the globin trypsin digest which eluted closely to the modified peptides were used as the “native reference peptide (NRP)” originally described by Ruse and coworkers for phosphopeptides.30 Using the peptide present in the protein digest (native peptide) as the reference peptide allows corrections for variations in the amount of protein used and in the

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peptide recovery during the digestion procedures. This relative quantification method permits comparison of the extent of modifications in a protein between samples and have been applied to various modifications.16,32-35 We used -Met-76 peptide as the reference peptide for modifications on -Cys-104 and -Cys-112 because their retention times are close. Because the content of methionine sulfoxide is substantial, the sum of peak areas of unmodified and sulfoxide of -Met-76 peptides was used the denominator. The native peptide of 18VNVDEVGGEALG30R in -globin is chosen as the reference peptide for -Cys-93 because it meets the criteria for being a reference peptide and it elutes ca. 2 minutes earlier than the sulfonic acid of the -Cys-93-containing peptide. REOMCO2 = ACO2 / (AMet + Asulfoxide) for -Cys-104 REOMCO3 = ACO3 / (AMet + Asulfoxide) for -Cys-104 and -Cys-112 REOMCO3 = ACO3 / (Aref + ACO3) for -Cys-93 Using this method, quantification of the extent of twelve sites and types modifications in a total of 37 samples (described below) is reproducible. The relative standard deviation (RSD) in triplicated experiments averages 12.3%, ranging from 5.0% to 18%. The RSD of the reference peptides averages 7%, indicating their consistent presence in the digest. Dose-Dependent Formation of Chlorinated Peptides. In order to identify the target peak in the complex chromatograms, the dose-response experiment was performed. In the digest of HOCl-treated hemoglobin, the peak with its area increases with increasing concentration of HOCl is the peak of interest. The identity of the peak eluting at 34.61 min is confirmed by its MS/MS spectrum. In hemoglobin incubated with HOCl, the peak area and hence the extents of chlorination at -Tyr-24 increased with increasing HOCl concentrations (Figure 2).

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Artifactual Formation of Methionine Sulfoxide. To evaluate the extent of artifactual methionine oxidation, such as during sample processing, trypsin digestion, and electrospray, the -Met-32-containing peptide MFLSFPTTK with [13C6]Leu was synthesized. After globin was isolated from blood, it was incubated with the surfactant SDS and heated briefly to denature the protein, followed by precipitation with acetone to remove the surfactant. If the isotope-labeled peptide was added in these steps, it would not be precipitated quantitatively with the denatured protein and cannot monitor the recovery properly. Hence, the isotope-labeled peptide was added to the denatured globin before trypsin digestion and analyzed by nanoLCNSI/MS/MS for the extents of methionine sulfoxide and sulfone formation. The synthetic [13C6]MFLSFPTTK itself contains 0.8% of methionine sulfoxide, presumably due to oxidation during the electrospray process as reported.36,37 In the three blood samples analyzed, the extents of methionine sulfoxide on the [13C6]MFLSFPTTK are ca. 1.1%, accounting for oxidation during digestion and electrospray process, but those on the unlabeled MFLSFPTTK range from 2.5% to 10.9% (Table 2). The results indicate that artifactual formation of methionine sulfoxide is only a small portion of the overall methionine sulfoxide measured and suggest that the extent of methionine sulfoxide formation can be a good index of endogenous oxidative stress in an individual. In all the samples examined, no methionine sulfone was detected. Extents of Modification in Hemoglobin Freshly Isolated from Human Blood. The extents of chlorination, nitration, and nitrosylation of tyrosine, along with oxidation of cysteine and methionine, residues were simultaneously measured in the trypsin digest of globin freshly isolated from human blood by nanoLCNSI/MS/MS under the SRM mode (Table S3,

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Supporting Information). Care has to be taken to isolate globin from fresh blood with the heme group detached. Detection of hemoglobin oxidation during clinical storage of red blood cells by mass spectrometry has been reported recently.38-40 This study included 19 T2DM patients and 18 nondiabetic individuals as the control group. Chlorination at -Tyr-24 was detected in all the samples. As shown in the representative nanoLC-NSI/MS/MS chromatograms, chlorinated -Tyr-24-containing peptide eluted 3.5 min later than the unmodified peptide but earlier than the nitrosylated and nitrated peptide (Figure 3A). To confirm the identity of these peaks, chromatograms of each modified peptide were confirmed using three different SRM transitions. The chromatograms for chlorinated -Tyr-24-containing peptide is shown in Figure 3B and those for the other eleven modified peptides are shown in Figure S2, Supporting Information. The MS/MS spectra of these modified peptides provide additional evidence for their identity (Figure S3, Supporting Information). Statistical Significance. The extents of hemoglobin chlorination at α-Tyr-24 and α-Tyr-42, nitration at α-Tyr-42, and oxidation at all three methionine residues are significantly higher in the 19 diabetic patients than in 18 normal individuals (p < 0.05) using the nonparametric Mann-Whitney U-test (Table 3). Among them, the p values of 0.0009 and 0.0008 for the extent of chlorination at α-Tyr-24 and nitration at α-Tyr-42 indicate the difference between the T2DM and the control groups is extremely significant (Figure S4, Supporting Information). Using the nonparametric Spearman correlation analysis of all the modifications with age, BMI, and HbA1c, the results reveal that seven out of the twelve sites and types modifications are associated with age significantly (p = 0.00010.0086) and that oxidation of the three methionine residues are associated with BMI extremely significantly (p =

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