Quantitative Mass Spectrometry Defines an Oxidative Hotspot in

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Quantitative Mass Spectrometry Defines an Oxidative Hotspot in Hemoglobin that is Specifically Protected by Haptoglobin Tatiana Pimenova,† Claudia P. Pereira,| Peter Gehrig,§ Paul W. Buehler,⊥ Dominik J. Schaer,*,| and Renato Zenobi*,† Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland, Center for Biologics Evaluation and Research (CBER), U.S. Food and Drug Administration (FDA), Functional Genomics Center Zurich, UZH/ETH Zurich, Switzerland, and Internal Medicine, University of Zurich, Switzerland Received March 19, 2010

The reaction of hemoglobin (Hb) with hydrogen peroxide (H2O2) results in free radicals generated at the heme iron, followed by radical transfer to the porphyrin/globin. In the present work, we employed isobaric tagging for relative and absolute quantification (iTRAQ) and a LC-MALDI-MS/MS-based proteomic approach to identify the extent of oxidative changes within tetrameric Hb and dimeric Hb-haptoglobin (Hb-Hp) complexes. Extensive oxidative modifications were found to be restricted to peptides containing RTyr42, βTyr145, and βCys93. The protein region composed of these peptides appears to define an area of oxidative activity within the Hb tetramer that extends across the critical R1β2/R2β1 interface. Extensive oxidative modifications occurring at βCys93 indicate that this surface amino acid is an important end point for free radical induced protein oxidation within Hb. Conversely when Hp 1-1 or 2-2 was complexed with dissociable Hb, oxidative changes in Hp complexed dimeric Hb were prevented. This protection was not observed in a stabilized tetrameric Hb, which displays a weak binding affinity for Hp. Therefore, dimerization of Hb and Hp binding may interfere with free radical translocation and play an important role in the overall antioxidant mechanism of Hp. Interestingly, the prevention of peroxide induced Hb amino acid oxidation in purified Hb-Hp1-1 and Hb-Hp2-2 was found to be equal, indicating a phenotype independent specificity in the process of oxidative protection. Taken together, these data suggest differences in oxidative modifications resulting from peroxide induced heme emanated free radical distribution in tetrameric compared to Hp1-1/Hp2-2 stabilized dimeric Hb. Keywords: hemoglobin oxidation • haptoglobin • quantification • iTRAQ • mass spectrometry

Introduction Oxidative modification of proteins is implicated in a number of diseases,1,2 such as Alzheimer’s disease,3-5 diabetes,6 atherosclerosis7 and cardiovascular disorders.8 Oxidative damage can cause irreversible structural changes in proteins leading to an alteration of their biological functions.9,10 Hemoglobin (Hb) is a particularly reactive and thus vulnerable protein that is released into extracellular milieus during diverse pathologic conditions, such as the hereditary hemolysis syndromes (i.e., sickle cell disease), malaria or atherosclerosis with intraplaque hemorrhage. Therefore, oxidation-induced structural changes in Hb could be directly involved in the pathogenesis of these and other diseases. * To whom correspondence should be addressed. Prof. Renato Zenobi, Department of Chemistry and Applied Biosciences ETH Zurich, CH-8093 Zurich, Switzerland, [email protected], and Dominik J. Schaer Internal Medicine, University Hospital, CH-8091 Zurich, [email protected]. † ETH Zurich. ⊥ U.S. Food and Drug Administration. § UZH/ETH Zurich. | University of Zurich. 10.1021/pr100252e

 2010 American Chemical Society

In the presence of H2O2, extracellular Hb is susceptible to heme iron and protein oxidation. H2O2 oxidizes deoxy-ferrous Hb (Fe2+) to generate the oxo-ferryl (Fe4+dO) state. In the case of the reaction with ferric Hb (Fe3+), a protein radical ( · Hb Fe4+dO) is formed as follows: Hb(Fe2+) + H2O2 f Hb(Fe4+dO) + H2O

(1)

Hb(Fe4+dO) + H+ f Hb(Fe3+) + OH-

(2)

Hb(Fe3+) + H2O2 f · Hb(Fe4+dO) + H2O

(3)

The nature of the protein-based radical in Hb has not been unequivocally revealed but the primary radical is assumed to be a heme iron derived neutral radical that can subsequently either dissipate into the aqueous medium or migrate to other amino acid, typically tyrosine residues, with final termination as oxidative protein modifications. One such radical translocation has been shown to occur between RTyr42 and βCys93 but other such pathways likely exist. Even at low concentrations Journal of Proteome Research 2010, 9, 4061–4070 4061 Published on Web 06/22/2010

research articles of H2O2, several amino acids within the β-globin chain (Trp15, Met55, Cys93 and Cys112) have been identified that become irreversibly oxidized.11 Additionally, extensive globin chain cross-links and protein-heme adducts form and together result in complete loss of hemoglobin structure. Haptoglobin (Hp) protects against the peroxidative effects of Hb in certain tissue compartments and seems to have a specific protective activity against structural Hb dimer oxidative modification during oxidative stress.12,13 Although much research has been done to characterize structural changes in oxidized human Hb, the extent of Hb oxidative modifications and the protective effect of Hp has not yet been quantitatively investigated. Therefore, no data exist that would allow us to model the spatial distribution of oxidative processes within tetrameric Hb and the Hb-Hp complex, respectively. A genetic haptoglobin polymorphism exists in humans that results in two different Hp phenotypes. Hp1-1 is a dimeric protein that can bind two Hb Rβ-dimers per molecule. Hp2-2 has a polymeric conformation with multiple (>3) binding sites. The two Hp phenotypes have been reported to have a different capacity to prevent hemoglobin-induced oxidative tissue damage with a generally higher risk of cardiovascular complications found in individuals with the Hp2-2 phenotype.14 It is, however, not known whether the inferior protection by Hp2-2 is a result of its smaller Hb binding affinity/capacity, the large molecular size that limits tissue penetration or whether Hp2-2 possesses a lesser ability to oxidatively protect Hb immobilized within the complex. Isobaric tagging for relative and absolute quantification (iTRAQ)15 in combination with tandem mass spectrometry (MS/MS) is an attractive technique that has been successfully applied in quantitative proteomics,16 for example to monitor differential protein expression and repression.16,17 In this study, we applied an iTRAQ labeling strategy to quantitatively analyze the spatial distribution of oxidative peptide modifications when Hb was reacted with H2O2 in the presence and absence of Hp. The results confirm and extend a model of Hb oxidative processes that involve a trans-subunit radical pathway which can be interrupted by sequestration of dimeric Hb within the Hb-Hp complex. To the best of our knowledge, it is the first time that an MS-based proteomic approach was employed to quantify oxidative modifications of proteins due to oxidative stress.

Experimental Section Preparation of Hemoglobin-Haptoglobin Complexes. An excess of Hb was incubated for 60 min at ambient temperature with either purified Hp1-1 or Hp2-2. 100 µL of each Hb was injected onto a BioSep-SEC-S 3000 (600 mm × 7.5 mm) size exclusion chromatography (SEC) column (Phenomenex, Torrance, CA) attached to a Waters 600 pump, Waters 2489 multiwavelength detector, and controlled by a Waters 600s controller using Empower2 software (Waters Corp., Milford, MA). The running buffer consisted of 0.1 M phosphate buffer, pH 7.0, pumped at a rate of 1.0 mL/min. Absorbance was monitored at 405 nm to detect heme-associated protein. SEC runs were terminated at 40 min, followed by a column equilibration of approximately 20 min between chromatography runs. Selected peaks for each Hb-Hp complex were collected over repeated injections to ensure the purity of 4062

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Pimenova et al. Hb-Hp complexes and rule out any interference from nonHp-bound Hb (see Supplemental Figure 1, Supporting Information). Preparation of Oxidized Samples (HbA0, rrXLHb, HbA0Hp1-1, HbA0Hp2-2 and rrXLHbHp1-1). In oxidation experiments, the following samples were used: highly purified hemoglobin, (HbA0 with 99% purity, prepared by Hemosol, Mississauga ON, Canada, obtained from U.S. FDA Center for Biologics Evaluation and Research); purified RR-cross-linked Hb (RRXLHb, prepared by U.S. Army, Washington, D.C., obtained from U.S. FDA Center for Biologics Evaluation and Research); haptoglobin of two phenotypes, Hp1-1 and Hp2-2 (Sigma-Aldrich Chemie GmbH, Buchs SG, Switzerland). Oxidation reactions were conducted as described earlier.18 Briefly, the solutions were prepared in 50 mM potassium phosphate buffer, pH 7.4, at a concentration of 250 µM and treated with H2O2, 30% w/w, (Sigma-Aldrich Chemie GmbH, Buchs SG, Switzerland). Heme/peroxide concentration ratios used were 1:1 and 1:10 in 1-mL volumes. Reactions were carried out at room temperature for 1 h, immediately followed by H2O2 removal using five buffer exchange steps with equal volumes of potassium phosphate buffer, pH 7.4, by filtering through 30kDa-cutoff centrifuge tubes (Centricon YM30, Millipore Corp, Bedford, MA). Samples were kept on ice or stored at -80 °C until analysis. iTRAQ Labeling. iTRAQ four-plex-reagents (Applied Biosystems, Foster City, CA) were used for quantification. Figure 1 depicts the workflow of the iTRAQ four-plex experiments. Table 1 lists the specific proteins used in the experiments (the colon (:) symbolizes noncovalent complexes, while the plus sign (+) symbolizes use of a protein mixture, without formation of a complex). In each iTRAQ four-plex experiment a set of four samples were used simultaneously. Unoxidized protein samples were used as a reference for the evaluation of the oxidation effect on the three test samples. Each iTRAQ set was treated the same way and analyzed 3 times. To 10 µL of each sample, containing 40 µg of hemoglobin in PBS buffer (concentration calculated for heme), 10 µL of dissolution buffer and 1 µL of 2% SDS (denaturant) was added. Following the manufacturer’s protocol (Applied Biosystems), the proteins were reduced with tris-(2carboxyethyl)phosphine hydrochloride (TCEPxHCl), blocked with methylmethanethiosulfonate (MMTS), and digested with modified porcine trypsin (Promega, Madison, WI) at an enzyme/ protein ratio of 1/50 (w/w) at 37 °C overnight. Afterward, iTRAQ reagents 114, 115, 116, and 117 were diluted in 70 µL of ethanol and added to the protein digests as depicted in Figure 1. The samples were incubated for 2 h at room temperature for iTRAQlabeling and then combined. Excess of iTRAQ reagents, SDS, TCEP and MMTS were removed by using a cation-exchange cartridge (ICAT-cartridge, Applied Biosystems) according to the manufacturer’s protocol. Then the samples were dried in a vacuum concentrator, resuspended in 40 µL of a 5% acetonitrile/0.1%TFA aqueous solution, and desalted with ZipTipC18 pipet tips (Millipore, Billerica, MA) according to the supplied user guide. The desalted samples were dried in a vacuum concentrator, redissolved in 10 µL of 5% acetonitrile/0.1%TFA water solution, and then subjected to liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (LC-MALDI-MS/MS) analysis. LC-MALDI-MS/MS Analyses. Peptide separation was performed on a chromatography system (Ultimate, Dionex - LC Packings, Sunnyvale, CA) equipped with a Probot MALDI

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Figure 1. Schematic workflow of four-plex iTRAQ experiments. Refer to Table 1 for identification of the various proteins and protein complexes.

spotting device. A volume of 5 µL of each sample was injected by using an autosampler (Famos, Dionex - LC Packings) and loaded directly onto a 75 µm × 150 mm reversed-phase column (PepMap 100, 3 µm; Dionex - LC Packings). Peptides were eluted at a flow rate of 300 nL/min by using the following

gradient: 0-10 min, 0% B; 10-75 min, 0-40% B; and 75-90 min, 40-60% B; 90-100 min, 60-100% B. Solvent A contained 5% acetonitrile and 0.1% trifluoroacetic acid in water, and solvent B contained 80% acetonitrile and 0.1% trifluoroacetic acid in water. For MALDI analyses, the peptides eluting from Journal of Proteome Research • Vol. 9, No. 8, 2010 4063

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Table 1. Types of Samples Used in Four-Plex iTRAQ Experiments

samples of the four-plex iTRAQ experiments

no.

1 2 3 4 5 6

reference sample no 0

test sample no 1

test sample no 2

test sample no 3

unoxidized protein

H2O2-oxidized protein

H2O2-oxidized protein complex

H2O2-oxidized protein complex

unoxidized[HbA0]

1:1[HbA0]

1:1[HbA0:Hp1-1]

1:1[HbA0:Hp2-2]

unoxidized[HbA0]

1:10[HbA0]

1:10[HbA0:Hp1-1]

1:10[HbA0:Hp2-2]

unoxidized[RRXLHb]

1:1[RRXLHb]

1:1[RRXLHb+Hp1-1]

1:1[RRXLHb+Hp2-2]

unoxidized[RRXLHb]

1:10[RRXLHb]

1:10[RRXLHb+Hp1-1]

1:10[RRXLHb+Hp2-2]

unoxidized[Hp1-1]

1:10[Hp1-1]

1:10[HbA0:Hp1-1]

1:10[HbA0:Hp2-2]

unoxidized[Hp1-1]

1:10[Hp1-1]

1:10[RRXLHb+Hp1-1]

1:10[RRXLHb+Hp2-2]

a Subscripts 1:1 and 1:10 correspond to a molar ratio of HbA0: H2O2. The colon (:) symbolizes noncovalent complexes, while the plus sign (+) symbolizes use of a protein mixture, without formation of a complex.

the column were directly mixed with a MALDI matrix solution (R-cyano-4-hydroxycinnamic acid, from Fluka) prepared at 3 mg/mL in aqueous solution of 70% acetonitrile, 0.1% trifluoroacetic acid, at a flow rate of 1.1 µL/min via a µ-Tee fitting. The matrix solution also contained neurotensin at a concentration of 125 pmol/mL (Sigma, St. Louis, MO) for internal calibration. The fractions were automatically deposited every 10 s onto a MALDI target plate (Applied Biosystems/MDS Sciex, Foster City, CA) using a Probot micro fraction collector. A total of 416 spots were collected from each HPLC run. Mass spectrometric analyses were carried out on a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems). The instrument was equipped with a Nd:YAG laser operating at 200 Hz. Mass spectra were automatically acquired in positive reflectron mode by accumulating data from 800 laser shots. First, spectra from peptide standards were used to update the instrument calibration. Mass spectra from all 416 LC-fractions were then recorded and internally calibrated based on the signal of the neurotensin peptide (m/z 1672.918). Spectral peaks that met the threshold criteria were included in the acquisition list for the MS/MS spectra. The following threshold criteria and parameter settings were used: mass range: 750 to 4000 Da; minimum signal-to-noise (S/N) for MS/MS acquisition: 100; maximum number of peaks/spot: 6. Collision-induced dissociation (CID) was performed at a collision energy of 1 kV and a collision gas pressure of approximately 2.5 × 10-6 Torr. During MS/MS data acquisition, a method with a “stop condition” was used. In this method, a minimum of 1000 shots (20 subspectra accumulated from 50 laser shots each) and a maximum of 2000 shots (40 subspectra) were allowed for each spectrum. The accumulation of additional laser shots was halted whenever at least 6 ion signals with a S/N of at least 60 were present in the accumulated MS/MS spectrum in the region from m/z 200 to 90% of the precursor ion mass. Data Evaluation. Data was analyzed by ProteinPilot software19 (version 2.0.1, Applied Biosystems) and searched against a human protein sequence database downloaded from ftp.ebi.ac.uk/pub/databases/SPproteomes (database release May 29, 2007; 38 041 protein sequences). The following search parameters were used: trypsin digestion, modifications of methylmethanethiosulfate-labeled (MMTS) cysteine, iTRAQ modifications of free amines at the N-terminus and of lysines, and iTRAQ modifications of tyrosine, serine and threonine amino acid residues. The peptides without any iTRAQ label at the N-terminus or at a lysine were excluded from the analysis. No normalization for iTRAQ ratios, such as BIAS correction, was applied. All possible biological modifications were also included in the search. The ProteinPilot cutoff score used was 1.3, which corresponds to a confidence limit of 95%. The relative quan4064

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titative data from iTRAQ-labeled tryptic peptides was derived from MS/MS spectra. The peak areas under the iTRAQ diagnostic ion signals at m/z 114.1, 115.1, 116.1, and 117.1 Da were divided by the peak area of a reference ion signal, which was selected from these four iTRAQ labeles depending on the experimental set. High-Mass MALDI Mass Spectrometry. Complexes of H2O2oxidized Hb (oxHb) and Hp1-1 as well as H2O2-treated Hb and Hp1-1 were analyzed by a MALDI-TOF mass spectrometer (Axima CFR, Shimadzu/Kratos Analytical, Manchester, U.K.) equipped with a high-mass detector (HM1, CovalX, Zurich, Switzerland). This allows sensitive analysis of macromolecules in the 10-1500 kDa range. MALDI instruments operated with standard detectors are often not capable to efficiently detect masses above 30-50 kDa.20 Our instrument was operated in linear positive mode by applying an acceleration voltage of 20 kV. Mass spectra were acquired by averaging 100 laser shots at different locations within a sample. Noncovalent protein complexes were stabilized with an amine-reactive homobifunctional cross-linking reagent, disuccinimidyl suberate (DSS, Pierce, Rockforf, IL). For stabilization, 1 µL of 4 mg/mL DSS in dimethylformamide was added to 10 µL of sample solution in the 1-5 µM concentration range. The samples were incubated at room temperature for about 2 h. For MALDI analysis, 1 µL of sinapinic acid, 10 mg/mL in 50% acetonitrile/0.1% trifluoroacetic acid in water, was mixed with 1 µL of the protein solution, and 1 µL was spotted onto the MALDI plate. The mass spectra were smoothed using instrument specific software (Kratos/Shimadzu).

Results and Discussion iTRAQ Quantification of Oxidized Amino Acids in Human Hb. The extent of oxidative modifications in human Hb induced by hydrogen peroxide in the absence and presence of haptoglobinwasinvestigatedusingiTRAQ-labelingandLC-MALDIMS/MS analysis. The experimental workflow is shown in Figure 1. Differential comparison of oxidized amino acids in Hb incubated with H2O2 in molar ratios of 1:1 and 1:10 (test sample 1) and in H2O2-treated complexes of [Hb-Hp1-1] and [Hb-Hp2-2] (test samples 2 and 3) was performed. Nonoxidized Hb was chosen as reference in all experiments. The four different samples were treated equally as described in the experimental part, were labeled with the four different iTRAQ reagents and pooled. During MS/MS analysis, the isobaric tags were cleaved and iTRAQ diagnostic ion signals showed up in the low-mass region at mass-to-charge (m/z) values of 114.1, 115.1, 116.1, and 117.1 Da. The ratios of the peak areas of these fragment ions relative to the 114.1 Da reference ion (corresponding to

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Oxidative Hotspot in Hemoglobin Protected by Haptoglobin

between different peptides/amino acids we used the oxidative loss of the nonmodified peptides to relatively quantify and compare the oxidative modifications of different peptides within Hb.

Figure 2. Grouped column graphs displaying iTRAQ ratios of tryptic peptides identified with oxidative modifications in the R- and β-chain of Hb with their corresponding error bars plotted on a logarithmic scale. The iTRAQ ratios were calculated for the Hb nonmodified peptides in the samples treated with H2O2 at a molar ratio of (A) 1:1 and (B) 1:10 relative to the same peptide in unoxidized HbA0. The bars therefore represent the oxidative loss of the respective tryptic peptides. (C) Corresponding amino acid numbers of the Hb peptides identified in the R- and β-chains. The bars shown in red depict the quantitatively most extensively modified peptides highlighted as an oxidative hotspot in Figure 4.

respective peptide quantity in nonoxidized Hb) provided quantitative information on the relative amount of a particular native or oxidatively modified peptide in H2O2-treated samples. The reference that we used in our experiments, nonoxidized Hb, is in fact slightly oxidized due to auto-oxidation processes.21 These auto-oxidative changes are minimal and this fact allows the identification and relative quantification of oxiditatively modified peptides. However, because the baseline concentration of oxidized residues must be assumed to be highly variable

The level of oxidative peptide modifications measured as an oxidative loss of unmodified peptides in Hb treated with H2O2 at a molar ratio of 1:1 (Figure 2A) is generally lower than when a ratio of 1:10 is used (Figure 2B), suggesting a direct relationship of heme redox cycling and peptide modifications. A wide range of different modifications can be identified in H2O2treated Hb (Table 2). In addition to the previously characterized amino acid modifications, we found hydroxylation of histidine (H2[O]), tyrosine (Y145[O]) and proline (P36[O]) in the Hb β-chain together with W37[Kyn], W37[O] and W37[O2] oxidation products of tryptophan 37. However, while these modifications are usually observed only in the context of relatively mild reduction in measurable unmodified peptides, the quantitatively most dramatic changes were observed for the β-chain peptides 83-104 and 133-146, and for the R-chain peptide 41-56. In case of β-chain peptide 83-104 the oxidative loss of the unmodified peptide was compensated by a relative increase in the corresponding peptide with a triple oxidation at βCys93 (cysteic acid) (Figure 3A). This modification was by far the most prevalent amino acid oxidation found in Hb reacted with H2O2 at 1:1 and 1:10 molar ratios, indicating that βCys93 serves as a primary radical sink during Hb peroxidative reactions. In case of the R-chain peptide 41-56 and β-chain peptide 133-146 we were not able to identify a simple amino acid oxidation or any other searchable modification that could compensate for the drastic oxidative loss of the native peptides, suggesting that more complex structural alterations occur in these two peptides. Interestingly, both peptides contain a potentially radical reactive Tyr and, in fact, electron paramagnetic resonance (EPR) analysis of RTyr42 mutant Hb as well as spin trapping experiments implied that this residue could be an essential part of a critical radical translocation pathway.22 We therefore assume that the two peptides participate in complex crosslinking reactions either with other peptides (i.e., by dithyrosine formation involving RTyr42/βTyr145) or with a structurally altered heme moiety. This interpretation is also well compatible with our previous observation of extensive intersubunit crosslinking that involves particularly Hb R-globin chains when reacting with H2O2.18 In Figure 4, the three most extensively modified peptides are projected in a Hb structural model. The clustering of these peptides and the putatively involved redoxactive amino acids identifies a novel oxidative hotspot in

Table 2. Oxidative Modifications Identified in Hb Following the Treatment with H2O2 Hb peptides

amino acid sequences

oxidative modifications

R_17-31 R_32-40 R_41-56 R_62-90 β_1-8 β_9-17 β_18-30 β_31-40 β_41-59 β_83-95 β_83-104 β_105-120 β_133-146

K.VGAHAGEYGAEALER.M R.MFLSFPTTK.T K.TYFPHFDLSHGSAQVK.G K.VADALTNAVAHVDDMPNALSALSDLHAHK.L -.VHLTPEEK.S K.SAVTALWGK.V K.VNVDEVGGEALGR.L R.LLVVYPWTQR.F R.FFESFGDLSTPDAVMGNPK.V K.GTFATLSELHCDK.L K.GTFATLSELHCDKLHVDPENFR.L R.LLGNVLVCVLAHHFGK.E K.VVAGVANALAHKYH.-

H20[O] M32[O] Y42[O] M76[O] H2[O] W15[Kyn], W15[O], W15[O2] no modification W37[Kyn], W37[O], W37[O2], P36[O], W37[O] M55[O] C93[O3] C93[O3] C112[O3] Y145[O] Journal of Proteome Research • Vol. 9, No. 8, 2010 4065

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Figure 3. (A) Bar graphs displaying iTRAQ ratios of the peptides β_83-104 and β_9-17 with oxidative modifications identified as a result of treatment with H2O2 in the molar ratios of 1:1 (light gray bars) and 1:10 (dark gray bars) relative to the same peptide in Hb not oxidized. The iTRAQ ratios are shown for both not modified (not mod) form of a peptide or MMTS-modified and with oxidative modifications. (B) MS/MS analysis of the tryptic peptide β_83-104_C93[O3] ) [3Ox] ) triple oxidation of HbA0 oxidized with a 10-fold molar excess of H2O2. The sequence of the peptide is shown with the resulting CID fragments for the b- and y-type ions. [IT4] indicates any of the 4-iTRAQ labels. The mass spectrum of the iTRAQ reporter ions is given in the inset, where the ions 114, 115, 116, and 117 correspond to unoxidized HbA0, HbA0_ox1:10, [HbA0:Hp1-1]_ox1:10 and [HbA0:Hp2-2]_ox1:10, respectively.

tetrameric Hb that extends across the R1β2/R2β1 (dimerdimer) interface. Preservation of Structural Hb Integrity within the Dimeric Hb-Hp Complex. We have recently described a protective effect of haptoglobin that helps to preserve Hb’s structural integrity during its reaction with H2O2.23 Studying oxidative changes in the dimeric Hb-Hp complex might therefore be a model to evaluate the role of subunit spanning radical translocations at the R1β2/R2β1 interface. We therefore carried out a quantitative iTRAQ analysis of oxidative modifications of amino acids in Hb when [Hb-Hp1-1] and [Hb-Hp2-2] complexes were formed and then exposed to H2O2. As shown in Figure 5, Hp completely prevents the oxidative damage of Hb amino acids as evidenced by the fact that no oxidative loss 4066

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of Hb peptides is observed when Hb is in complex with Hp during H2O2 reaction, even at a 10-fold molar excess of the oxidant. We expected that the dimeric Hp1-1 and polymeric Hp2-2 might have divergent protective effects since the two Hp phenotypes have different Hb binding affinities and different physiological effects. Hp1-1 is assumed to protect more effectively against oxidative stress induced by extracellular hemoglobin than the polymeric Hp phenotype Hp2-2.24 We therefore compared quantitatively the extent of oxidation and protection, respectively, when Hb was bound to Hp1-1 or Hp2-2 before exposure to H2O2. In these experiments we found no quantitative difference in oxidative loss of unmodified Hb peptides within Hb-Hp1-1 and Hb-Hp2-2 complexes. The

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Figure 4. Three-dimensional structure of the oxidative hotspot at the Hb R1β2-interface. The modifiable amino acids within these peptides (βCys93, βTyr145 and RTyr42) are highlighted. The R-globin chain is in light gray, the β-globin chain is in dark gray, and heme molecules are shown in green as stick models. The PDB structure 1O1K was imported to Chimera (UCSF) to generate the image and highlight amino acids.

Figure 5. Quantitative evaluation of Hp1-1 (black circles) and Hp2-2 (white circles) protective capacity from oxidation of Hb in the Hb-Hp complexes treated with a 10-fold molar excess of H2O2. Both Hp1-1 and Hp2-2 protect Hb from oxidation.

reason for this finding could be the fact that we used HPLCpurified complexes of [Hb-Hp1-1] and [Hb-Hp2-2] to avoid the potentially confounding effect of the uneven binding capacity of the two Hp phenotypes (capacity Hp2-2 < capacity Hp1-1). Additionally, high-mass MALDI-TOF mass spectrometric analysis25 revealed that the [Hb-Hp1-1] high affinity complex was preserved when the oxidative insult attacked the preformed complex. However, Hb-Hp complex formation was completely abolished when the tetrameric Hb was first exposed to H2O2 (Figure 6A). After exposure of the preformed [Hb-Hp1-1] complex to H2O2, the signals for the specific complexes corresponding to Hp1-1 and one Hb dimer (Hb(Rβ)-Hp1-1) and two HbA0 dimers (Hb(Rβ)2-Hp1-1) at 123.6 kDa and 154.3 kDa, respectively, were detected. A peak at 138.7 kDa represents different stoichiometries of Hb R/β-globin chains incorporated in Hb-Hp complex and the peak at 16.1 kDa corresponds to free R/β-chains of Hb. In contrast, no binding of Hp1-1 to H2O2-treated Hb was observed (Figure 6B). Only two pro-

Figure 6. High-mass MALDI mass spectra. (A) H2O2-treated [HbA0: Hp1-1] complexes at 123.6, 138.7, and 154.3 kDa, which represent different stoichiometries of Hb R/β-globins within the complexes (top) DSS-stabilized, (bottom) without DSS stabilization. (B) H2O2-treated HbA0 at 66.5 kDa and Hp1-1 at 91.3 kDa. No [HbA0:Hp1-1] complexes were detected. The mass shifts observed between DSS-stabilized and without stabilization for the same species are due to covalent modification of proteins with DSS reagent.

nounced peaks corresponding to unbound Hp1-1 at 91.3 kDa and to the tetrameric form of oxidized Hb (oxHb(Rβ)2) at 66.5 kDa were detected. The less abundant peak at 33.5 kDa is the doubly charged form of the 66.5 kDa species. To prevent dissociation of noncovalent complexes during high-mass MALDI MS analysis a cross-linking reagent, DSS, was employed. Figure 6 shows DSS-stabilized mixtures of Hb and Hp1-1 (upper spectra traces) along with the spectra without DSSstabilization (lower spectra traces). The mass shift that was observed for the same species in the mass spectra after DSSstabilization corresponds to DSS molecules covalently attached to the proteins. These MALDI data indicate that Hp does not only prevent oxidation of specific amino acids but effectively preserves overall structural integrity of hemoglobin. Specificity in Hp Shielding against Peroxidative Modifications in Native Hb. To examine the specific role of dimeric Hb-Hp complex formation in oxidative protection we used a chemically modified RR-cross-linked tetrameric Hb (RRXLHb) Journal of Proteome Research • Vol. 9, No. 8, 2010 4067

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Pimenova et al. destructive radical delocalization pathway in Hb that involves generation of protein based radicals at the R-globin heme group, temporal stabilization on RTyr42 and subsequent passage to the β-globin chain amino acids, mainly βCys93. This model has initially been derived from EPR experiments and both radicals on RTyr42 and βCys93 could subsequently being trapped by the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).28-30 The close proximity of RTyr42 to heme and βCys93 and the fact that RTyr42 and βCys93 peptides are by far the most extensively modified peptides, as shown here, support the role of these residues as primary radical scavengers within Hb. The role of other, more distant β-globin residues remains to be cleared but it might be that these amino acids (i.e., Trp15, Trp37) form escape pathways that become relevant after complete oxidation of βCys93 to its triple oxidation product cysteic acid.

Figure 7. Oxidative modification pattern and lack of Hp protection in the β-chain of tetramer stabilized Hb (RRXLHb). The log of the abundance ratios of unmodified β-chain peptides in native and H2O2 (1:10) treated Hb are shown. Each symbol represents one specific peptide in RRXLHb or HbA0, respectively. (A)Very similar oxidative loss in RRXLHb peptides (x-axis) versus HbA0 (y-axis) peptides (slope close to 1). (B) When Hp is added before the hemoglobins were exposed to H2O2, the oxidative loss of peptides in HbA0 is close to 0, while the peptides in RRXLHb are still oxidatively modified. In the presence of Hp HbA0 peptides are completely preserved, while the oxidative loss remains significant in the tetramer stabilized Hb. The most extensively modified and protected peptides are highlighted in red (βTyr145 containing peptide) and green (βCys93 containing peptides).

that can not dissociate into Rβ-dimer under physiologic conditions. We previously found that the tetramer stabilization in this Hb drastically reduces high affinity Hb-Hp complex formation.26,27 While the general pattern and extend of amino acid oxidations in RRXLHb was similar to HbA0, Hp did only provide very limited protection against H2O2-induced structural changes in RRXLHb (Figure 7). The high affinity interaction between the two proteins is therefore essential for Hb’s oxidative protection by Hp. Within the R-globin chain loss of the Tyr42 containing peptide, presumably by unspecified cross-linking-reactions, was the most notable protein modification detected after Hb was reacted with H2O2. In contrast, specific amino acid oxidation events dominate the structural modification of the Hb β-globin chain. Our quantitative study is the first evidence that βCys93 is the most vulnerable amino acid with an exceptionally high ratio of oxidative modification in the form of cysteic acid compared to oxidative modifications of other amino acid residues. However, as compared to other oxidizable β-subunit residues βCys93 was also the most protected when Hb was reacted with H2O2 within the Hp complex. The quantitative data established in this study are compatible with a proposed 4068

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We have previously shown that heme’s reaction with H2O2 follow the same kinetics of redox cycling from Fe3+ f Fe4+ whether Hb is bound to Hp or not.23 Also, certain ligand binding as well as autoxidation kinetics are only minimally altered whether Hb is free or within a Hb-Hp complex.13 Therefore, the production of potentially damaging radicals can be presumed to be similar in Hb and Hb-Hp. The comparable ligand binding and reaction kinetics of Hb and Hb-Hp also argue against drastic structural rearrangements of Hb upon binding to Hp. How then are the amino acids and overall structural integrity of Hb protected when Hb is exposed to H2O2 within the Hp complex? Considering the available evidence, several mechanisms could be involved in this protection. (1) A critical event during complex formation might be that the Hb tetramer is forced into a dimeric state during Hp complex formation.31 In contrast to Hb alone, which exists as a heterodimer (tetramer/dimer) equilibrium, exclusively dimers of Hb bound to Hp are present in a solution of Hb-Hp complexes. As shown in Figure 4, the proposed RTyr42f βCys93 radical migration pathway extends across the R1β2 (or R2β1) interface. Therefore, forced dimerization of Hb could interrupt this pathway and may thereby prevent the reaction of vulnerable β-chain amino acids with RTyr42-based radicals. (2) When trapped within the complex, Hb protein radicals could migrate to Hp itself. Possible structural imprints that could point to a relevant redirection of the heme derived radicals to Hp would be amino acid oxidations in Hp peptides or covalent cross-links forming between Hb amino acids and Hp (i.e., TyrTyr cross-links). In our iTRAQ studies we did not consistently detect any prevalent Hp peptide oxidation, although the protein sequence coverage of Hp was not optimal (37%) (Supplemental Figure 2, Supporting Information). In contrast, strong evidence for covalent HbHp cross-links can be found in our own and other’s published work.23,32 After the reaction of Hb-Hp complexes with H2O2, a new band appears in reducing/ denaturing SDS-PAGE gels that has a molecular weight compatible with a protein species consisting of a Hb globin chain covalently linked to the Hp β-subunit. In both studies the same band was consistently detected by different antibodies directed against Hb or Hp. Although the specific sites of these crosslinks have not been revealed yet, the findings suggest that Hp plays a more direct role in Hb protection than just forcing the Hb tetramer to dimerize. (3) A third mechanism could involve stabilization of protein based radicals in the Hb-Hp complex. In contrast to Hb that follows a self-destructive peroxidase reaction path; certain peroxidase enzymes contain proteinlinked heme and defined mechanisms to contain toxic oxidative

research articles

Oxidative Hotspot in Hemoglobin Protected by Haptoglobin species. Although stabilization of protein radicals within the Hb-Hp complex seems to be an attractive possibility, there is so far only very limited evidence to support this mechanism. Kapralov et al. showed that the magnitude of the low-temperetaure EPR detectable protein radical was higher in Hb-Hp compared to Hb alone during H2O2 reaction.32 However, neither the localization of the radical nor its fate have been examined in these studies.

Conclusions In the current study, we quantitatively defined the spatial distribution of oxidative damage in tetrameric Hb following its reaction with H2O2. In contrast to earlier structural studies of Hb damage under oxidative conditions, our quantitative mass spectrometry approach allowed us for the first time to localize the most extensive radical damage to a confined protein region containing RTyr42, βTyr145 and βCys93. Spatial analysis of our data indicates that this oxidatively active protein region expands across the R1β2/R2β1 interfaces of tetrameric Hb. Additional oxidations at His2, Trp15, Pro36, Trp37, Met55 and Cys112 amino acid residues in the Hb β-chain could be confirmed but these changes are quantitatively minor. Therefore, while βCys93 seems to be a primary radical destination, these less modified amino acids might be end points of secondary or alternative escape radical pathways that become only relevant after complete oxidation of βCys93. In the presence of H2O2, abundant changes within the RTyr42, βTyr145 and βCys93 amino acid regions appear to define an area of oxidative modification that is completely protected when dimeric Hb is bound in a complex with either of the two major human haptoglobin phenotypes.

Acknowledgment. We acknowledge Abdu Alayash, CBER, FDA, Bethesda for providing the purified RR-crosslinked Hb. This work was supported by the Swiss National Science Foundation grant 31-120658 (D.J.S.). The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Supporting Information Available: Supplemental Figure 1. Separation and purification of HbA0 complexed with Hp1-1 (top) andHp 2-2 (bottom). All hemoglobin-haptoglobin (Hb-Hp) complexes isolated on a BioSep S-3000 SEC colum (7.5 × 600 mm). Supplemental Figure 2. Grouped column graphs, plotted on a logarithmic scale, display iTRAQ ratios of tryptic peptides in haptoglobin (Hp) with their oxidative modifications identified as a result of treatment with H2O2 in the molar ratios of 1:1 (light gray bars) and 1:10 (dark gray bars) relative to the same peptide in Hp not oxidized. For each peptide the iTRAQ ratios are shown for both not modified (not mod) form of a peptide or MMTS-modified and with oxidative modifications. Supplemental Figure 3. MS/MS analysis of the Hb tryptic peptides (A) β_133-146_Y145[O])[Oxi] and (B) R_41-56_Y42[O])[Oxi]. The sequences of the peptides are shown with the resulting CID fragments for the b- and y-type ions. [IT4] indicates any of the 4-iTRAQ labels. The mass spectra of the iTRAQ reporter ions are given in the insets, where the ions 114, 115, 116, and 117 correspond to unoxidized HbA0, HbA0_ox1:10, [HbA0:Hp1-1]_ox1:10 and [HbA0:Hp2-2]_ox1: 10, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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