Mass Tagging Approach for Mitochondrial Thiol Proteins - Journal of

Yu, L. R.; Conrads, T. P.; Uo, T.; Issaq, H. J.; Morrison, R. S.; Veenstra, T. D. J. Proteome Res. 2004, 3, 469−477. [ACS Full Text ACS Full Text ],...
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Mass Tagging Approach for Mitochondrial Thiol Proteins Kevin Marley,† Duane T. Mooney,† Gretchen Clark-Scannell,† Tony T.-H. Tong,† Jeffrey Watson,‡ Tory M. Hagen,§ Jan F. Stevens,| and Claudia S. Maier*,† Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon 97331, Linus Pauling Institute and Department of Biochemistry & Biophysics, Oregon State University, Corvallis, Oregon 97331, and Department of Pharmaceutical Sciences and the Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331 Received March 28, 2005

A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d0 and IBTP-d15. The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d0/d15-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications. Keywords: Isotope-coded labeling • mass spectrometry • mitochondria • thiol modifications

Introduction Thiol proteins represent an important subclass of the mitochondrial proteome. For instance, thiol proteins are involved in redox signaling,1 the permeability transition,2 and apoptosis.3 Many constituents of the respiratory chain complexes are thiol proteins.4 Because of the nucleophilicity of the sulfhydryl group, thiol proteins are potential targets of oxidative stress-related modification reactions. Covalent modifications of thiol proteins that can be attributed to oxidative stress include protein glutathionylation, their oxidation to sulfenic acid and higher oxidation states, Michael-type adductions with reactive lipid peroxidation products, and S-nitrosylation.5,6 Oxidative damage to proteins has been implicated in degenerative diseases and aging.7 Recently, Lin et al. reported the use of (4-iodobutyl)triphenylphosphonium (IBTP) for the specific modification of mitochondrial protein thiols in order to investigate the effects of oxidative stress on the thiol redox status.8 The lipophilic phosphonium cation is accumulated in the mitochondria due * To whom correspondence should be addressed. Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331. Phone: (541) 737-9533. Fax: (541) 737-2062. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Biochemistry & Biophysics. § Linus Pauling Institute and Department of Biochemistry & Biophysics. | Department of Pharmaceutical Sciences and the Linus Pauling Institute. 10.1021/pr050078k CCC: $30.25

 2005 American Chemical Society

to the mitochondrial membrane potential. IBTP reacts with surface-exposed protein thiols in a SN2-type alkylation reaction to form a stable thioether bond (Figure 1A). Using twodimensional electrophoresis and an IBTP-specific antibody these authors were able to monitor the change of the thiol redox state of distinct mitochondrial proteins following oxidative stress.8 In the present work, we describe a mass tagging approach based on the IBTP-chemistry that is designed for use in mass spectrometry-based quantitative proteomics studies of mitochondrial thiol proteins. To identify and to quantify mitochondrial thiol proteins by a mass spectrometry-based approach, we introduce a mass-coded IBTP-analogue. Similar in idea to the ICAT approach described by Gygi et al.,9 we use the isotopomers, IBTP-d0 and IBTP-d15, as the ‘light’ and the ‘heavy’ labeling reagents, respectively. Here, we discuss the mass spectrometric characteristics of the IBTP reagent as a selective and stable isotope-coded label for thiol proteins and the applicability of the IBTP-labeling approach for the identification and relative quantification of thiol proteins from rat heart mitochondria.

Experimental Section Materials. E. coli thioredoxin and proteomics-grade trypsin were purchsed from Promega (Madison, WI). Tris(2-carboxyJournal of Proteome Research 2005, 4, 1403-1412

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an enzyme to substrate (E:S) ratio of 1:50 (w/w). Digestion was stopped by adding acetic acid to final concentration of 10%. Prior to mass spectrometric analysis samples were stored at -20 °C. IBTP-Labeling of Lactoglobulin. Lactoglobulin (55 µM, 1 mg/mL in phosphate buffer) was either incubated with 1.6 mM IBTP-d0 or 1.6 mM IBTP-d15 for 30 min at room temperature in the dark. The IBTP-d0 and -d15 stock solutions (33 mM) were prepared in 100 mM phosphate buffer (pH 8.2) containing 50% acetonitrile. Aliquots of the ‘light’ and ‘heavy’ reaction mixtures were mixed in different ratios to obtain samples that contained protein-d0 (light)/protein-d15 (heavy) ratios ranging from 0.1 to 5. For example, 25 µL (i.e. 1.35 nmol lactoglobulin) aliquots of lactoglobulin-d0 reaction mixture were incubated with different amounts of lactoglobulin-d15 reaction mixtures (5250 µL, i.e., 270 pmol to 13.5 nmol lactoglobulin). Samples were dialyzed against 50 mM ammonium acetate, lyophilized to dryness and stored at -20 °C. Lyophilized samples were dissolved in 50 µL 100 mM NH4HCO3, pH 8.2, and digested using a 1:50 (w/w) ratio of trypsin to lactoglobulin. Digestion was performed over a period of 6 h at 37 °C. Digestion was stopped by adding TFA to a final concentration of 0.1%.

Figure 1. Modification reaction of protein thiolates with (4iodobutyl)-triphenylphosphonium (IBTP). A, Modification reaction of protein thiolates with IBTP; B, Structure model of IBTP with the van der Waals surface area depicted by dots. The distances between atoms are given in Å.

ethyl)phosphine hydrochloride (TCEP), Bradford and bicinchonic acid (BCA) protein assay reagent kits were from Pierce Biotechnology (Rockford, IL). Iodoacetamide, lactoglobulin and Glu-fibrinopeptide B were purchased from Sigma-Aldrich (St. Louis, MO). Synthesis of IBTP-d0 and -d15. IBTP-d0 was synthesized by reacting 1,4-diiodobutane (5-fold molar excess) with triphenylphosphine at 100 °C under nitrogen in the dark for 1.5 h following the procedure described by Lin and co-workers.8 This procedure was also used to prepare IBTP-d15 from 1,4-diiodobutane and tri(phenyl-d5)phosphine. IBTP-Labeling of E. coli Thioredoxin. Solutions of E. coli thioredoxin were prepared at concentrations of 85 µM (1 mg/ mL) in 100 mM phosphate buffer at pH 8.4. TCEP was added to a final concentration of 0.5 mM (150 µg/mL). The reduction of the disulfide bond was complete after 30 min at room temperature. The IBTP-d0 stock solution (33 mM, 19 mg/mL) was prepared in 100 mM phosphate buffer at pH 8.2 containing 50% acetonitrile. IBTP labeling was performed in the dark at room temperature for 2 h. The concentration of IBTP-d0 during alkylation was approximately 2 mM. To block the Cys-35 thiol group of E. coli thioredoxin, carboxyamidomethylation was performed for 60 min at room temperature in the dark by adding a 25-fold excess of iodoacetamide (54 mM, 10 mg/mL) in 100 mM phosphate buffer (pH 8.4). Excess reagent was removed by dialysis against 50 mM ammonium acetate. The IBTP-labeled and carboxyamidomethylated thioredoxin was stored at -20 °C. For digestion, IBTP-tagged protein in 125 mM NH4HCO3 (0.8 mg/mL) was incubated overnight at 37 °C with trypsin using 1404

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IBTP-Labeling of Mitochondrial Thiol Proteins. Rat heart mitochondria were isolated according to Suh et al.10 The IBTPd0 and -d15 stock solutions (33 mM) were prepared in 50 mM potassium phosphate buffer (pH 8.2) containing 30% acetonitrile. Aliquots of frozen mitochondria were suspended in 10 mM potassium phosphate buffer (pH 8.2) containing 250 mM sucrose and treated with IBTP-d0 and -d15 for 30 min at 37 °C. The final concentration of IBTP-d0 or -d15 was approximately 1 mM. Mitochondria were disrupted by several freeze-thaw cycles. Soluble and insoluble protein fractions were obtained by centrifugation. Protein concentration was determined by using the BCA and Bradford method. The soluble mitochondrial proteins were digested in 125 mM NH4HCO3 with trypsin using an E:S ratio of 1:30 (w/w) overnight at 37 °C. Acetic acid was added to a final concentration of 10% to stop digestion. NanoLC-ESI-MS/MS Analysis. Proteolytic digests of IBTPlabeled proteins were analyzed by nanoLC-ESI-MS/MS using a quadrupole orthogonal time-of-flight mass spectrometer (QTOF Global Ultima, Micromass/Waters, Manchester, UK). Reverse-phase nanoLC was performed using a capillary HPLC (CapLC, Waters, Milford, MA) with flow split to approximately 300 nL/min for the analytical separation on a 10 cm × 75 µm in-house packed Jupiter C5 or C18 (Phenomenex Inc., Torrance, CA) picofrit column (New Objectives, Woburn, MA). Peptide samples were injected onto a 5 mm × 0.32 mm C4 or C18 trap cartridge (Waters, Milford, MA) at a flow rate of 12 µL/min to concentrate and desalt the samples. After 6 min, the trap cartridge was automatically switched in-line with the analytical column directly coupled to the mass spectrometer. Solvent A was 3% acetonitrile containing 0.1% formic acid and 0.005% TFA. As solvent B, 95% acetonitrile containing 0.1% formic acid and 0.005% TFA was used. The following gradient for peptide separation was employed: 0-5 min, 3% solvent B; 5-40 min 3 to 40% solvent B, 40-50 min 40 to 70% solvent B, 50-60 min 70 to 95% solvent B. The electrospray ion source was operated in the positive ion mode with a spray voltage of 3.5 kV. The data-dependent MS/ MS mode was used with a 0.5 s survey scan and 2.5 s MS/MS scans on the three most abundant ion signals in the MS survey scan, with previously selected m/z values being excluded for

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Mass Tagging of Mitochondrial Protein Thiols

60 s. The collision energy for MS/MS (25 to 65 eV) was dynamically selected based on the charge state of the ion selected by the quadrupole analyzer (q1). Mass spectra were calibrated using fragment ions of Glu1-fibrinopeptide B (MH+1570.6774 Da, monoisotopic mass). Capillary LC-MALDI-MS/MS Analysis. Peptide samples were loaded onto a 5 × 0.32 mm C18 trap cartridge (Waters, Milford, MA) at a flow rate of 15 µL/min. After 10 min, the trap cartridge was automatically switched in-line with the analytical 150 × 0.3 mm Symmetry C18 column using a Waters capLC system coupled to a prototype, pneumatically assisted MALDI target spotter. Peptides were eluted with a gradient from 5% to 90% B over 60 min using as solvent A, 5% acetonitrile containing 0.1% TFA, and as solvent B, 90% acetonitrile containing 0.1% TFA. The flow rate was 3 µL/min. The column effluent was mixed with R-cyano-4-hydroxycinnamic acid (2 mg/mL in 50% acetonitrile containing 0.1% TFA) via a Teejunction. The matrix/effluent solution was then periodically collected onto a stainless steel target plate. MALDI-MS/MS analysis was performed on an ABI 4700 Proteomics Analyzer with TOF/TOF optics equipped with a 200 Hz frequency-tripled Nd:YAG laser operating at a wavelength of 355 nm (Applied Biosystems, Inc. Framingham, MA). Mass spectra were acquired in the reflector mode with an accelerating voltage of 20 kV. For MS/MS experiments, the collision energy, which is defined by the potential difference between the source acceleration voltage (8 kV) and the floating collision cell (7 kV), was set at 1 kV. The precursor ion was selected by operating the timed gate window with 3-10 Da. Gas pressure (air) in the collision cell was set to 6 × 10-7 Torr. Fragment ions were accelerated by 15 kV into the reflector. Data were acquired in a mass range of m/z 700-4000 with external calibration using AB’s 4700 calibration mixture consisting of the following peptides (in brackets the monoisotopic mass of the singly protonated ion is given in Da): des-arg1-bradykinin (904.4681), angiotensin I (1296.6853), Glu1-fibrinopeptide B (1570.6774), and ACTH 18-39 (2465.1989). Mass Spectral Data Processing and Database Searching. For ESI-MS/MS data, MassLynx 4.0 was used for processing raw MS/MS data, generating peak list files and determining peak areas from extracted ion chromatograms. In addition, the expression analysis module of ProteinLynx 2.1 (Micromass/ Waters Corp.) was used occasionally for data analysis, in particular for aiding in the extraction of the d0- and d15-peptide pairs. However, to be accepted as d0/d15 peptide pair the spectra were visually inspected to determine if the ion peaks of the peptide pairs had the same charge state, if the peptide isotopomers eluted within the expected time window under consideration of the isotope retention time shift and if the mass difference of the peptide isotopomers was 15 Da, i.e., ∆m ) 7.5 (2+), 5.0 (3+), or 3.75 (4+). MALDI-MS/MS data were processed with GPS explorer 2.0 for creating Mascot-searchable files. For protein identification, MS/MS data were searched using the Mascot search engine (Matrix Science, London, UK) against the mammalian NCBInr/MSDB database (Taxonomy rodentia) with oxidized Met (147.04 Da, monoisotopic mass), IBTP-d0 and -d15-labeled Cys (420.15 Da, and 435.25 Da, respectively) as variable modifications. Searches were done with initial mass tolerance of 1.5 Da in the MS mode and 0.25 Da in the MS/MS mode. Peptide ions score below 30 were not accepted. In addition, tandem mass spectra of IBTP-labeled peptides were

visually inspected for preferred fragmentation patterns such as N-terminal cleavage to a proline residue. Molecular Modeling. Modeling was performed with InsightII software (Accelrys, San Diego, CA). The butyltriphenylphosphonium (BTP) and the carboxyamidomethyl (CAM) moieties were generated from standard InsightII libraries, and a formal charge of +1 was added to the phosphorus atom of the BTP molecule. X-ray crystal structures of thioredoxin (PDB accession code 2TRX) and creatine kinase (PDB accession code 1CRK) were retrieved from the Protein Data Bank. BTP and CAM were covalently attached to Cys-32 and Cys-35 of thioredoxin, respectively. BTP was covalently attached to Cys-199 of each of the four monomers of the creatine kinase tetramer. To simplify the minimization and dynamics calculations for the BTP adduct of creatine kinase, only residues 189-209, 10 residues N- and C-terminal to the modified cysteine, were allowed to move. Both the thioredoxin and creatine kinase structures were subjected to 5 rounds of 100 steps of energy minimization and 2 rounds of 1000 steps of molecular dynamics simulation at 300 K using the AMBER force field. The dimensions and surface area for IBTP (Figure 1B) were obtained using VOIDOO (Uppsala Software Factory). The surface area was calculated using a 1.4 Å radius sphere and the van der Waals radii for all atoms. All other parameters used were from VOIDOO standard libraries.

Results Mass Spectrometric Properties of IBTP-Tagged Peptides. To study the mass spectrometric properties of IBTP-tagged peptides, the oxidoreductase thioredoxin was reduced with TCEP and reacted with IBTP at pH 8 for 30 min at room temperature. Carboxyamidomethylation was used to block remaining thiol groups. Following digestion with trypsin, the sample was analyzed by µLC-ESI-MS/MS on the quadrupole orthogonal time-of-flight mass spectrometer (qTOF). The IBTPlabeled peptide showed intense signals at m/z 1177.5 and m/z 785.4 for the doubly and triply charged ions, respectively. It is important to note that the butyltriphenylphosphonium moiety introduces a fixed positive charge in the peptide (without protonation). This has to be considered for the determination of the peptide mass. From the [M+H]2+ and [M+2H]3+ ion signals the mass of the IBTP-labeled peptide was determined to be 2355.1 Da which agrees well with the calculated peptide mass after accounting for the IBTP modification (+ 317 Da, monoisotopic mass) and carboxyamidomethylation (+ 57 Da, monoisotopic mass). The collision-induced dissociation (CID) spectrum recorded for the triply charged precursor ion [M+2H]3+ with m/z 785.4 showed an intense doubly charged y-ion series ranging from y62+ to y172+ and a less intense singly charged y-ion series ranging from y6 to y11 (Figure 2A). In addition, the y4-ion at m/z 461.2 indicated that Cys-35 was modified by carboxyamidomethylation (160 Da, monoisotopic mass for the S-carboxyamidomethyl cysteine residue). The y5-ion was not observed, but the y6-ion at m/z 1067.5 was, indicating that the Cys-32 residue was modified by IBTP (420.16 Da, monoisotopic mass for the butyl triphenylphosphonium-modified cysteine residue). The y6-fragment ion retained the mass tag during the low energy CID experiment. No side-chain specific ions of the butyltriphenylphosphonium moiety were observed. The absence of side-chain fragmentation eliminates complications in the interpretation of low energy MS/MS spectra and allows for automated database searches based on MS/MS data. Journal of Proteome Research • Vol. 4, No. 4, 2005 1405

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Figure 3. LC-ESI-MS full scan mass spectrum (A) and MS/MS spectra (B) of the lactoglobulin peptide aa 165-178 labeled with IBTP-d0 and IBTP-d15.

Figure 2. Mass spectrometric properties of the IBTP-tagged tryptic peptide aa 19-36 derived from the model protein E.coli thioredoxin. A, Low energy-CID MS/MS spectrum of the peptide aa 19-36 acquired on an ESI qTOF-type instrument. The butyltriphenylphosphonium tag causes charge-directed backbone fragmentation. No side chain fragmentation of the BTP moiety was observed. B, High Energy-CID MS/MS spectrum of the peptide aa 19-36 obtained on a MALDI-TOF/TOF instrument. Butyltriphenylphosphonium-specific side-chain fragment ions are visible in the low m/z-range.

To study the mass spectrometric properties of the IBTPlabeled peptide in MALDI-TOF/TOF collision induced dissociation (CID) experiments, the tryptic digest of IBTP-treated and carboxyamidomethylated TRX was separated on a C18 capillary column, and the effluent directly spotted onto a MALDI target after online mixing with R-cyano-4-hydroxycinammic acid. The MS survey analysis yielded the complete set of expected tryptic peptides. The IBTP-labeled peptide gave rise to an intense molecular ion peak at m/z 2354.1. The MALDI-TOF/TOF instrument uses high energy (1 kV) for CID experiments. The MS/MS analysis of the IBTP-labeled peptide yielded specific side-chain fragment ions of the butyltriphenylphosphonium moiety at m/z 262.3, 317.4, and 351.4 with high intensities, but peptide backbone fragment ions were observed only to a limited extent (Figure 2B). Identification and Quantification Accuracy of IBTP-Labeled Peptides. To test the accuracy of identification and quantification, we analyzed tryptic digests of IBTP-d0 and IBTP-d15labeled lactoglobulin (lg). An 18 µM bovine lactoglobulin stock solution in phosphate buffer (pH 8) was prepared. Subse1406

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quently, aliquots of the lactoglobulin solution were treated separately with IBTP-d0 and IBTP-d15. The labeled protein mixtures were combined, dialyzed, proteolyzed with trypsin and analyzed by LC-ESI-MS/MS on a qTOF instrument operated in the data-dependent acquisition mode. IBTP-labeled peptide pairs are recognizable by their mass difference of 15 Da. The mass-to-charge (m/z) difference between the isotopically distinct peptides depends on the charge state, and is either 7.5 (2+), 5.0 (3+) or 3.75 (4+). Figure 3 shows an example of a mass-tagged peptide pair. Collision induced dissociation of the triply charged molecular ion with m/z 659.0 (d0-analog) and 664 (d15-analog) yielded MS/MS spectra exhibiting intense doubly charged y-fragment ion series, i.e., y62+ to y132+, whereby the y2+ ions that originated from the d15-analogue were consistently shifted by 7.5 Da. Similarly, the y6 and y7 ions that were observed for both isotopomers were shifted by 15 Da in the MS/MS spectrum of the triply charged molecular ion of the d15-isotopomer. Database searching using Cys-BTP-d0 and -d15 as variable modifications identified the isotopomeric peptide analogues as the tryptic C-terminal peptide of lactoglobulin, namely LSFNPTQLEEQCHI, encompassing residues 165 to 178 (annotation according to SwissProt entry P02754) with Ile-178 being the C-terminal amino acid residue of lactoglobulin. Because of the thiol specificity of the IBTP reagents, it can be assumed that Cys-176 was modified by IBTPd0 and -d15, respectively. In Figure 4, the LC-MALDI-MS/MS analysis of the same peptide, aa 165-178 LSFNPTQLEEQCHI, is illustrated. In MALDI-TOF mass spectra usually singly charged peptides are observed. As such, the respective isotopomeric peptide pair can be easily analyzed by its mass difference of 15 Da. Tandem mass spectrometry of the d0-peptide at m/z 2975.96 using MALDI-TOF/TOF high energy collision induced dissociation yielded y-type fragment ions. Fragment ions of the butyltri-

Mass Tagging of Mitochondrial Protein Thiols

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Figure 4. LC-MALDI MS/MS measurement of the relative abundances of peptides tagged with IBTP-d0 and IBTP-d15. A, Pairs of IBTP-tagged peptides can be identified by a 15 Da-mass difference in the mass spectrum. B, MALDI-MS/MS analysis of the IBTP-d0-tagged peptide. Mascot identified this peptide as the partial sequence aa 165-178 of lactoglobulin. Specific fragment ions of the isotope-coded probe in the low mass range facilitate the identification of tagged peptides.

phenylphosphonium moiety due to side-chain fragmentation are visible in the low m/z-range. To assess the accuracy of quantification of the IBTP mass tagging approach, IBTP-d0 and IBTP-d15 labeled protein mixtures were combined in different ratios, dialyzed and digested with trypsin. For best quantification results, the qTOF instrument was operated in the MS survey mode and reconstructed ion chromatograms of the d0- and d15-tagged peptide were used in the analysis. Due to the isotopic shift during chromatography, the d15-tagged peptide eluted approximately 5-10 s before the d0-analogue. Differences in retention times of ICAT-tagged peptides were also observed in the applications of the first generation ICAT reagents.9 The differences in retention times for ICAT-labeled peptides using the deuterium-substituted ICAT reagent were also in the range of several seconds.11 Quantitation for the lactoglobulin-derived peptide aa 165-178 was demonstrated by determining the d0/d15-tagged peptide ratios from the respective peak areas obtained from extracted ion chromatograms. The experimentally obtained peak area ratios d0/d15 were then compared with the expected ratios based on the protein amounts mixed. The doubly and triply charged ions were evaluated. Linearity was observed over a 50fold range for both charge states (Figure 5A). In LC-MALDI MS/MS experiments, the automated target spotter functions essentially as miniaturized fraction collector. This eliminates chromatographic isotopic shift in retention times and suggests that MALDI-MS/MS can be particularly promising for determining accurate, relative quantities of

Figure 5. Relative peak area d0/d15-ratio measurements of the IBTP-labeled peptide encompassing the residues 165-178 of lactoglobulin (lg) by LC-ESI-MS (A) and MALDI-MS (B). Solutions containing known amounts of lactoglobulin were incubated with either IBTP-d0 or IBTP-d15. Aliquots of the ‘light’ and ‘heavy’ reaction mixtures were mixed in different ratios to obtain samples that contained protein-d0 (light)/protein-d15 (heavy) ratios ranging from 0.1 to 5. Specifically, 25 µL (i.e., 1.35 nmol lactoglobulin) aliquots of lactoglobulin-d0 reaction mixture were incubated with different amounts of lactoglobulin-d15 reaction mixtures (5 to 250 µL, i.e., 270 pmol - 13.5 nmol lactoglobulin). After dialysis, lyophilization and trypsination digests were analyzed by LC-ESI-MS (A) and MALDI-MS (B). For both mass spectrometric methods plotting the experimentally obtained d0/ d15 peptide ion ratios against the expected d0/d15 ratios gave a linear response over a 50-fold concentration range.

isotopically labeled peptide pairs. The area of the isotopic ion peaks for the labeled peptide was used to obtain the relative abundance d0/d15 ratios. The results indicated that MALDIMS analysis allowed accurate estimation of relative peptide abundances over a 50-fold range (Figure 5B). Selective Labeling of Mitochondrial Thiol Proteins. To demonstrate the feasibility of using stable isotope-coded IBTP reagents to selectively target and potentially quantify mitochondrial thiol proteins, we treated mitochondrial protein preparations containing variable amounts of protein separately with excess IBTP-d0 and IBTP-d15. Alkylation reactions were performed in phosphate buffer pH 8.5 for 30 min at 37 °C. The IBTP-d0 and IBTP-d15-treated mitochondrial protein preparations were combined and the alkylation reagents were removed Journal of Proteome Research • Vol. 4, No. 4, 2005 1407

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Table 1. IBTP-tagged Peptides Identified by LC-ESI-MS/MS identified protein

Dihydrolipoamide succinyltransferase (DLST) Creatine kinase Aconitase Isocitrate dehydrogenase Acetyl-CoAC-acyltransferase

sequence of IBTP-tagged peptidea

ions scoreb

gi/266684

EAQNTC*AMLTTFNEVDMSNIQEMR

86

gi/92102 gi/10637996 gi/34857317 gi/66509

LIDDHFLFDKPVSPLLTC*AGMAR VGLIGSC*TNSSYEDMGR SSGGFVWAC*K VVGYFVSGC*DPAIMGIGPVPAITGALK

61 49 44 41

accession no.

a The asterisk / marks the cysteine residue which is the likely modification site by IBTP. b The highest ions score observed in three LC-ESI-MS/MS experiments is listed.

Figure 6. Protein identification and quantification approach for mitochondrial thiol proteins using the stable isotope-coded probe IBTP-d0/d15. A, Full scan (300-2000 m/z (mass-to-charge) mass spectrum at a particular time point) of the LC-ESI-MS experiment. Peptide pairs are identified by their mass difference of 15 Da. The m/z difference between the peptides is dependent on the charge state and typically is either 7.5 (2+), 5.0 (3+), or 3.75 (4+). B, An expanded view of the full scan mass spectrum shows the ion signal for an IBTP-tagged peptide pair. C, Peptide identity was established in a separate LC-ESI-MS/MS experiment, in which the qTOF instrument was operated in the MS to MS/MS switching mode and set to acquire MS/MS data of peptides that were part of an “inclusion list”, a list of parent ions of IBTP-d0-tagged peptides. The MS/MS data depicted were searched against the MSDB database (taxonomy: rodentia) and identified as peptide aa 221-243 of creatine kinase, sarcomeric mitochondrial.

by dialysis. Proteolysis with trypsin was performed overnight at 37 °C. The ratio of the IBTP-d0 and IBTP-d15 labeled peptides was determined from their peak areas obtained in the case of ESI-MS from selected ion chromatograms. Peptide identification was achieved by MS/MS-based sequence tagging. The mitochondrial peptide digests were analyzed by both LC-ESI-MS/MS and LC-MALDI-MS/MS. Combining the search results of both techniques these experiments yielded 207 peptides. These peptides were observed across three experiments. The peptide data were compiled and this resulted in the identification of 51 proteins (Supplementary Table 1 in the Supporting Information). The peptide per protein average for this set of proteins identified by both techniques was 4.1 ()207/51). The relative high value obtained for peptide per protein average reflects that for most protein identifications at least 2 peptides had to be identified with high confidence, 1408

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i.e., the ion score was set to 30 as threshold for accepting a peptide as identified. However, the LC-MALDI-MS/MS analyses did not result in the identification of IBTP-labeled peptides. A likely reason for this observation is that the high energy-CID experiments used in the TOF/TOF instrument causes side chain-fragmentation of IBTP-modified cysteine residues which results in tandem mass spectra that are less useful for Mascot searches. About 14 IBTP-labeled peptide pairs were detected by LC-ESI-MS/MS analysis. Mass-tagged peptide pairs that gave ion signals that were intense enough for unambiguous MS/MS identification are listed in Table 1. The annotated tandem mass spectra are available as Supporting Information (Supplementary Figure 1). The tandem mass spectrum of a peptide (1031.4, 3+) from dihydrolipoamide succinyltransferase yielded the highest ion score of the IBTP-tagged peptides, namely 86, and this spec-

Mass Tagging of Mitochondrial Protein Thiols

trum was the best scoring spectrum obtained from all IBTP tagged-peptides (Supplementary Figure 1A). Interesting features of this spectrum include a doubly charged b-ion series (b82+ to b142+, and b202+) and an extended y-ion series (y2 to y16). Double charging of b-ions was observed for the fragment ions encompassing the butyltriphenylphosphonium-tagged cysteine (BTPCys) residue, whereas singly charged y-ions were observed for the peptide sequence C-terminal to the BTP-Cys residue. This observation of doubly charged b-ions, starting with b82+, is consistent with the notion that the introduction of a fixed charge by the butyltriphenylphosphonium moiety promotes formation of doubly charged fragment ions by charge retention. The tandem mass spectrum of the peptide (719.6, 4+) from creatine kinase (Figure 6C and Supplementary Figure 1B in the Supporting Information) is dominated by an intense doubly charged y-ion series encompassing y72+ to y182+ which includes the BTP-Cys residue. A short b-ion series (b5 to b6) is also observed. The ions score for this spectrum is 61. Interestingly, the presence of proline residues in this peptide promotes the fragmentation N-terminal to the respective proline residues giving rise to the relatively intense fragment ions y102+ and y132+. Note, KP is not a cleavage site for trypsin. The collision-induced fragmentation of peptide (702.3, 3+) from aconitase yielded doubly charged y-ions ranging from y112+ to y162+ and singly charged y-ion ranging form y4 to y10. Intriguingly, all doubly charged y-ions encompass the BTP-Cys residue, whereas the singly charged y-ions were observed for fragment ions that originated from the partial sequence Cterminal to the BTP-Cys residue. The ions score for this spectrum is 49 (Supplementary Figure 1C in the Supporting Information). Tandem mass spectrometric fragmentation of the peptide (679.3, 2+) from isocitrate dehydrogenase resulted in an almost complete y-ion series (y2 to y8), a short doubly charged y-series (y42+ to y82+ except y72+) and a few b-ions (b2, b4 to b6); the ions score for this spectrum is 44. In this peptide, the BTPtagged cysteine residue is directly adjacent to the C-terminal Lys residue. This proximity may cause charge competition between the butyl triphenylphosphonium moiety and the -amino group of the Lys residue which may explain why in this spectrum the singly charged y-ion series dominates (Supplementary Figure 1D in the Supporting Information). The tandem mass spectrum of the peptide (983.5, 3+) from acetyl-CoA C-acyltransferase exhibits a short b-ion series (b3 to b5), an extended y-ion series (y8 to y15, except y9) and a doubly charged y-ion series encompassing y202+ to y252+ except y242+ (Figure 7 and Supplementary Figure 1E in the Supporting Information). The ions score of this spectrum is 41. Intense y8- and y10-ions are indicative of the preferred fragmentation of the amide bond N-terminal to the respective proline residues. Again, double charging of y-ions is only observed for fragment ions that include the BTP-Cys-residue. For determining the relative peptide abundances of IBTPtagged peptide pairs, LC-ESI-MS experiments were performed on mitochondrial protein extracts labeled either with IBTP-d0 or IBTP-d15. The reaction mixtures were combined so that after mixing the protein concentration ratio of the IBTP-d0/IBTPd15-treated protein extracts was approximately 2:1. For determining the isotopomeric ratios of IBTP-tagged peptide pairs, ion traces of LC-ESI-MS analyses (i.e., full scan mass spectra) were evaluated rather than the ion traces extracted from datadependent acquisitions. LC-ESI-MS data were required for using ProteinLynx Global server’s protein expression software

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Figure 7. Identification (A) and relative quantification (B) of the IBTP-tagged peptide aa 279-305 of acetyl-CoA C-acyl transferase by LC-ESI-MS/MS. Two mitochondrial protein preparations containing equal amounts of protein were treated with excess IBTP-d0 and IBTP-d15, respectively. After dialysis and trypsination, the mixture was analyzed by LC-ESI-MS/MS.

module to aid in the extraction of isotopomeric peptide pairs. The final positive identification of an isotopomeric peptide pair was done visually by confirming that the charge state was the same for each isotopomeric peptide and that the mass difference was 15 Da under consideration of the charge state, i.e., ∆(m/z) ) 7.5 (2+), 5.0 (3+), or 3.75 (4+). The ratios of d0- to d15-labeled peptide pairs were determined using MassLynx software by calculating the ratios according to the area of the extracted ion chromatogram of each labeled peptide. Peak area ratios for five identified mitochondrial peptides are listed in Table 2. The average ratios of these d0/d15-tagged peptide pairs obtained from three LC-ESI-MS experiments range from 1.51 ( 0.02 for the peptide from isocitrate dehydrogenase to 1.93 ( 0.05 for the peptide from creatine kinase. The overall average ratio for the mitochondrial peptides identified was 1.72 and the standard deviation was 0.19. The overall variability in peptide quantitation for these peptides was 11%. The variability of the LC-ESI-MS analyses, evaluated for each individual peptide pair, was the highest for the peptide from acetyl-CoA C-acyltransferase, namely 7.8%. To illustrate the generally good quantitation reproducibility for the same peptide the mass Journal of Proteome Research • Vol. 4, No. 4, 2005 1409

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Table 2. Peptide Peak Area Ratios d0- and d15-labeled Peptides Determined by LC-ESI-MS of Mitochondrial Protein Extracts Labeled with IBTP-d0 and IBTP-d15, Mixed and Trypsinateda

IBTP-tagged peptides fromb

m/z values of d0-/d15-isotopomer (charge state)

DLST CK Aco ICDH ACoAAT

1031.4/1036.4 (3+) 719.6/723.4 (4+) 702.3/707.3 (3+) 679.3/686.8 (2+) 983.5/988.5 (3+)

peak area ratios (d0/d15) run 1 run 2 run 3 average ( SD

1.82 1.98 1.49 1.50 1.90

1.92 1.90 1.63 1.53 1.79

1.81 1.87 1.46 1.51 1.63

1.85 ( 0.06 1.93 ( 0.05 1.53 ( 0.09 1.51 ( 0.02 1.77 ( 0.14

are highly susceptible to oxidative modifications. We introduced a mass spectrometry-based approach using the thiolspecific reagents IBTP-d0 and IBTP-d15 as stable isotope-coded probe. IBTP forms stable thioether bonds with sulfhydryl groups,8 making our approach a potentially useful tool for probing the redox proteome. The surface accessibility and the microenvironment affect the reactivity of the cysteine thiol functionality toward the alkylation reagent.

Discussion

For example, thioredoxin has two cysteine residues at position 32 and 35 in its active site, i.e., the “thioredoxin fold” encompassing the sequence Cys-X-Y-Cys. Cys-32 was preferentially found to be modified by IBTP under the conditions used. One likely reason for the predominant alkylation of Cys32 is the well-documented increased reactivity of Cys-32 toward electrophiles due to its uncommonly low pKa value of ∼7 compared to other cysteine thiols, which is caused in part by electrostatic interactions that favor the thiolate anion form.18,19 To get a first idea concerning the steric requirements of the IBTP reagents toward surface-exposed thiols, a structure model of IBTP was constructed (Figure 1B). A model was made for the modified thioredoxin in which the butyltriphenylphosphonium and carboxyamidomethyl moieties were covalently attached to Cys-32 and Cys-35, respectively. The model, based on the X-ray structure of reduced thioredoxin, was subjected to molecular dynamics and energy optimization using the AMBER force field (Figure 9). The dimensions of the butyltriphenylphosphonium moiety (Figure 1B) were compatible with the fully solvent-exposed orientation of the Cys-32 thiol. In contrast, the Cys-35 thiol was found to be accessible to the sterically less demanding carboxyamidomethyl moiety but not to the bulky butyltriphenylphosphonium group.

Protein thiols play a key role in redox reactions in mitochondria and represent an important group of proteins that

For some of the mitochondrial proteins from which IBTPlabeled peptides were detected information is available regard-

a The IBTP-d0-labeled protein extract was combined with the IBTP-d15labeled extract so that the d0/d15 ratio should be approximately 2:1. b DLST, dihydrolipoamide succinyltransferase; CK, creatine kinase; Aco, aconitase; ICDH, isocitrate dehydrogenase; ACoAAT, Acetyl-CoA C-acyltransferase.

spectra of the d0/d15 pair of the peptide aa 379-395 of aconitase from three consecutive LC-ESI-MS runs are depicted in Figure 8. For this peptide the variability was 5.9%. Factors that likely affect the quantitation accuracy of isotopomeric peptide pair ratios are (a) the ionization efficiency for the IBTP-labeled peptides which is influenced by the chemical composition of the individual peptides, (b) ion suppression effects caused by coeluting peptides, and (c) the solvent microenvironment in which the peptides elute. It is anticipated that the accuracy of determining relative isotopomeric peptide ratios can be further improved by including more sophisticated chromatographic separations for minimizing the coelution of nontagged peptides and the overlapping of peptide signals of nontagged and tagged peptides.

Figure 8. Reproducibility of relative peak area measurements illustrated for the peptide aa 379-395 of aconitase. A, Reconstructed ion chromatograms for a d0- and d15-tagged peptide pair that was identified as peptide aa 379-395 of mitochondrial aconitase. Due to the isotopic shift the d15-tagged peptides elutes approximately 0.15 min before the d0-analogue. B, Full scan mass spectra of three consecutive LC-ESI-MS runs of the same mitochondrial peptide preparation. The m/z-region of the d0/d15 peptide pair is shown. 1410

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Figure 9. Structure and model of the model protein E. coli thioredoxin. X-ray structure of reduced E. coli thioredoxin highlighting the surface accessibility of the thiol group of Cys32 compared to the thiol group of Cys-34. Ribbon model of IBTPtagged E. coli thioredoxin in orange superimposed on the ribbon model of E. coli thioredoxin in yellow. The butyltriphenylphosphonium (BTP) moiety and the modified cysteine residue are colored in blue. The model was constructed by covalently linking the positively charged BTP moiety to Cys-32 and the CAM moiety to Cys-35 and energy-minimized using the AMBER force field. The steric requirements of the BTP moiety determine which of the Cys residues of the thioredoxin fold is alkylated. This is consistent with the observation that IBTP alkylation occurred exclusively on Cys-32. The RMS deviation was calculated to be 1.89 Å which indicates a significant change in thioredoxin’s backbone.

ing the presence of reactive thiol groups. For instance, creatine kinase (CK; EC 2.7.3.2) was recently identified by mass spectrometry as an oxidation-sensitive protein in the brain of ApoEknockout mice,12 and was found to be oxidized in the brains of human subjects with Alzheimer’s disease.13 In our study, the peptide encompassing Cys-238 in rat mitochondrial creatine kinase, was found to be labeled by IBTP. The respective cysteine residue in rabbit skeletal muscle creatine kinase was recently described to be sensitive to oxidation by H2O2.14 Similarly, Furter et al. identified the corresponding cysteine residue in chicken creatine kinase as redox-sensitive.15 In Figure 10, the

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Figure 10. Structure and model of creatine kinase (chicken). BTPtagged creatine kinase. The residues 189-209 which were used for the energy minimization and dynamics are shown in pink and the solvent-exposed cysteine residue and the BTP moiety are shown in blue. The ribbon model of creatine kinase shown in blue is superimposed on the ribbon model of BTP-tagged creatine kinase shown in green. The residues 189-209 of the BTPtagged creatine kinase are shown in light pink. The same residues of the untagged protein are colored dark pink. Cys-199 is shown in blue. The calculated RMS deviation is 0.327 Å indicating little change has occurred in the backbone of the protein. The “bulkiness” of the BTP moiety is well tolerated by the microenvironment of the respective cysteine residue.

X-ray structure of mitochondrial creatine kinase (chicken) is displayed and the respective partial sequence encompassing the residues 182 to 204 is marked. In this structure, the thiol group of the Cys-residue is solvent-exposed and the microenvironment would allow the bulky butyltriphenylphosphonium moiety. A recent paper by Benderdour et al. describes the inactivation of mitochondrial NADP+-isocitrate dehydrogenase by 4-hydroxynonenal adduct formation which seems to involve a Cys-residue near the substrate binding site.16 The identification of the IBTP-tagged peptide of isocitrate dehydrogenase is in accord with a previous report in which the Cys residue in the peptide SSGGFVWACK was found to be highly susceptible to Journal of Proteome Research • Vol. 4, No. 4, 2005 1411

research articles alkylation by N-ethylmaleimide in the absence of substrate but seemed well protected in the presence of isocitrate. It was concluded that the Cys residue in SSGGFVWACK may have a catalytic role, most likely in strengthening the binding of Mn2+ in the presence of isocitrate.17 Several recent reports underline the sensitivity of aconitase to ROS and its concomitant loss of activity.18-20 For aconitase several inactivation pathways have been proposed. Dependent on the source of oxidative stress, key events that lead to enzyme inactivation and degradation include the release of the labile iron Fe-R from the [4Fe-4S]2+ cluster under formation of the [3Fe-4S]1+ cluster and cluster disassembly, post-translational modifications, e.g., protein carbonylation, and S-oxygenation.18 Interestingly, the IBTP-labeled residue of aconitase is a cysteine residue (Cys-385) which is involved in the ligation of the cubane Fe-S cluster in the porcine enzyme X-ray structure. We may therefore speculate that this particular cysteine residue might be also highly susceptible to modification reactions. Taken together, we have described a mass tagging approach for mitochondrial thiol proteins based on the stable isotopecoded thiol-specific alkylation reagents IBTP-d0 and IBTP-d15. The feasibility of using stable isotope-coded IBTP reagents, as an analytical tool for identifying protein thiols and determining their relative abundances, was demonstrated by using single protein models as well as a protein extract obtained from rat heart mitochondria. The mass spectrometric properties of the stable-isotope coded IBTP probes facilitated the identification of IBTP-tagged peptides. In low energy collision-induced dissociation experiments, IBTP-labeled peptides resulted in tandem mass spectra that were suitable for automated database searches. Measurements of the relative d0/d15 peptide abundances were precise as judged by an average quantitation variability of 11%. The reactivity of IBTP is apparently biased toward highly solvent-exposed thiols due to the bulky triphenyphosphonium moiety. This is not perceived necessarily as a disadvantage, because one can hypothesize that those cysteine thiols would be preferred targets of oxidative stressmediated modifications. Because of the lipophilic cationic structure of the IBTP reagents, the described mass tagging approach potentially provides an opportunity for mass spectrometry-based identification and relative quantification of modification-prone protein thiols in respiring mitochondria which may open new opportunities in mitochondrial redox proteomics. Abbreviations: BTP, butyltriphenylphosphonium; CAM, carboxyamidomethyl; CID, collision induced dissociation; ESI, electrospray ionization; LC, liquid chromatography; MALDI, matrix assisted laser desorption; MS, mass spectrometry; MS/ MS, tandem mass spectrometry; ICAT, isotope-coded affinity tag; IBTP, (4-iodobutyl)triphenylphosphonium; lg, lactoglobulin; RMS, root mean square; ROS, reactive oxygen species; TFA, trifluoroacetic acid.

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Acknowledgment. This study was supported by the National Institute on Aging (AG025372), the American Heart Association (no. 0460023Z), the Medical Research Foundation of Oregon and by the National Institute of Environmental Health Sciences (P30 ES00210). The authors wish to acknowledge the Mass Spectrometry facility and the Molecular Structure and Interactions facilities and services core of the Environmental Health Sciences Center at Oregon State University. Supporting Information Available: Table showing the mitochondrial proteins identified by LC-ESI-MS/MS and LC-MALDI-MS/MS; Figure displaying the annotated MS/MS spectra of IBTP-labeled peptides from cardiac mitochondrial protein extracts. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ghezzi, P.; Bonetto, V. Proteomics 2003, 3, 1145-1153. (2) Kanno, T.; Sato, E. E.; Muranaka, S.; Fujita, H.; Fujiwara, T.; Utsumi, T.; Inoue, M.; Utsumi, K. Free Radic. Res. 2004, 38, 2735. (3) Ghafourifar, P.; Klein, S. D.; Schucht, O.; Schenk, U.; Pruschy, M.; Rocha, S.; Richter, C. J. Biol. Chem. 1999, 274, 6080-6084. (4) Jha, N.; Jurma, O.; Lalli, G.; Liu, Y.; Pettus, E. H.; Greenamyre, J. T.; Liu, R. M.; Forman, H. J.; Andersen, J. K. J. Biol. Chem. 2000, 275, 26096-26101. (5) Stadtman, E. R. Ann. N. Y. Acad. Sci. 2001, 928, 22-38. (6) Levine, R. L.; Stadtman, E. R. Exp. Gerontol. 2001, 36, 1495-1502. (7) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239-247. (8) Lin, T. K.; Hughes, G.; Muratovska, A.; Blaikie, F. H.; Brookes, P. S.; Darley-Usmar, V.; Smith, R. A.; Murphy, M. P. J. Biol. Chem. 2002, 277, 17048-17056. (9) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (10) Suh, J. H.; Heath, S. H.; Hagen, T. M. Free Radic. Biol. Med. 2003, 35, 1064-1072. (11) Yu, L. R.; Conrads, T. P.; Uo, T.; Issaq, H. J.; Morrison, R. S.; Veenstra, T. D. J. Proteome Res. 2004, 3, 469-477. (12) Castegna, A.; Thongboonkerd, V.; Klein, J.; Lynn, B. C.; Wang, Y. L.; Osaka, H.; Wada, K.; Butterfield, D. A. J. Neurochem. 2004, 88, 1540-1546. (13) Aksenov, M.; Aksenova, M.; Butterfield, D. A.; Markesbery, W. R. J. Neurochem. 2000, 74, 2520-2527. (14) Sethuraman, M.; McComb, M. E.; Heibeck, T.; Costello, C. E.; Cohen, R. A. Mol. Cell Proteomics 2004, 3, 273-278. (15) Furter, R.; Furter-Graves, E. M.; Wallimann, T. Biochemistry 1993, 32, 7022-7029. (16) Benderdour, M.; Charron, G.; DeBlois, D.; Comte, B.; Des Rosiers, C. J. Biol. Chem. 2003, 278, 45154-45159. (17) Smyth, G. E.; Colman, R. F. J. Biol. Chem. 1991, 266, 14918-14925. (18) Bulteau, A. L.; Ikeda-Saito, M.; Szweda, L. I. Biochemistry 2003, 42, 14846-14855. (19) Sipos, I.; Tretter, L.; Adam-Vizi, V. J. Neurochem. 2003, 84, 112118. (20) Das, N.; Levine, R. L.; Orr, W. C.; Sohal, R. S. Biochem. J. 2001, 360, 209-216.

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