Analysis of protein phosphorylation by mass spectrometry

217Review Article

From the Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan,Taiwan.Received: Jun. 28, 2007; Accepted: Aug. 31, 2007Correspondence to: Prof. Pao-Chi Liao, Department of Environmental and Occupational Health, College of Medicine, NationalCheng Kung University. No. 138, Shengli Rd., North District, Tainan City 704, Taiwan (R.O.C.) Tel.: 886-6-2353535 ext. 5566; Fax: 886-6-2743748; E-mail: [email protected]

Analysis of Protein Phosphorylation Using Mass Spectrometry

Hsin-Yi Wu; Pao-Chi Liao, PhD

Protein phosphorylation has been known to be a pivotalmodification regulating many cellular activities and functions.Except for several conventional techniques, mass spectrome-try-based strategies are increasingly considered as vital toolsthat can be utilized to characterize phosphorylated peptides orproteins. In this article, we summarized currently availablemass spectrometry-based techniques for the analysis of phos-phorylation. Due to the low abundance of phosphopeptides,enrichment steps such as specific antibodies, immobilizedmetal affinity chromatography, and specific tags are crucial fortheir use in detection. Since the non-specific binding of theenrichment techniques are constantly of major concerns, phos-phatase treatment, neutral loss scan, or precursor ion scanenable the recognition of the phosphopeptide signals. In addi-tion, quantitative methods including isotope labeling and masstags are also discussed. Phosphoproteome analysis seems to provide elucidation of signalingnetworks and global decipherment of cell activities, which require powerful analytical meth-ods for complete and routine identification of the phosphorylation event. Despite thatnumerous approaches have been exploited, comprehensive analysis of protein phosphoryla-tion remains a challenging task. With the progressively more improvements of instrumentsand methodologies, we can foresee the implementation of a comprehensive approach for theanalysis of phosphorylation states of proteins. (Chang Gung Med J 2008;31:217-27)

Key words: phosphorylation, mass spectrometry, IMAC, phosphoproteome, quantitative analysis

Protein phosphorylation, an essential post-transla-tional modification, affects most cellar activities

including signal transduction, gene expression, cellcycle progression and other biological functions.(1,2)

Conventionally, radioactive 32P is introduced into cel-lular proteins via labeled ATP to trace phosphoryla-tion. Fractionation techniques, such as high-perfor-mance liquid chromatography and one or two dimen-sional gel electrophoreses are utilized subsequentlyfor the detection of the radioactive proteins. The

localization of the phosphorylation sites are achievedusing Edman degradation.(3) Although this techniquehas demonstrated its capability of analyzing proteinphosphorylation on some cases, it can be laborious,time consuming and Edman degradation may fail ifthe N-terminus of the peptide is blocked. Mass spec-trometry has been regarded as a powerful tool notonly for the identification of proteins but also theanalysis of post-translational modifications. Itstremendous accuracy of mass measurement suggests

Prof. Pao-Chi Liao

Chang Gung Med J Vol. 31 No. 3May-June 2008

Hsin-Yi Wu, et alProtein phosphorylation analysis

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the presence of protein modifications which mayresult in specific mass differences between modifiedand unmodified proteins. A number of researchershave featured the use of mass spectrometry in proteinphosphorylation analysis.(4-8) Nevertheless, someintrinsic characters of phosphopeptides have hin-dered the analysis by mass spectrometry. First, thestoichiometric level of phosphoprotein may be verylow. Second, phosphopeptides tend to have relativelylow ion abundance and suppression effect especiallyin the presence of non-phosphorylated peptides.Third, phosphate groups on phosphoserine and phos-phothreonine are labile, which could decompose dur-ing improper sample preparation or peptide fragmen-tation. Many efforts have been made to improve theanalysis of protein phosphorylation, as shown in Fig.1 which will be discussed in this report.

Enrichment methodsAntibody

Antibodies are frequently used to recognize spe-cific proteins. For phosphoproteins, a more generallyuseful tool would be amino-specific antibodies beingraised against phosphorylated proteins/peptides.Those site-specific antibodies can be utilized as priorenrichment (e.g. immunoprecipitation) of phosphory-lated species from complex samples. There are sev-eral commercially available antibodies that are ableto bind to phosphotyrosine, phosphoserine, and phos-

phothreonine residues. However, phosphotyrosine-specific antibodies are considered a relatively effi-cient way for analyzing tyrosine phosphorylation andhave demonstrated several successes in characteriz-ing tyrosine phosphorylation events.(9-11) The lack ofexcellent antibodies have been known to be the limi-tation of analyzing serine- and threonine-phosphory-lated proteins. Yet in 2002, Grønborg and hiscoworkers worked out a new set of antibodies direct-ed against phosphoserine and phosphothreonineresidues. These antibodies demonstrated the capabili-ties of enriching phophoserine- and phosphothreo-nine-containing proteins using a global approach.(12)

Chromatographic and affinity tag enrichment

As mentioned before, phosphopeptides are oftenof low abundance and their detection can be inter-fered with by the existence of non-phosphorylatedpeptides. Therefore, the complicated peptide mixturesamples call for procedures for extraction and enrich-ment of phosphopeptides before further MS analysis.The most widely used method for selectivity isimmobilized metal affinity chromatography (IMAC).This technique incorporates metal ions, usually Fe3+,Ga3+ or others (e.g. Zr4+), and bind them to a chelat-ing group. Phosphopeptides are bound due to theaffinity between the metal ions and phosphategroups.(13,14) The releasing of bound peptides can beachieved using a high pH or phosphate buffer. Eventhough the IMAC technique can be used successfullyduring both on-line and off-line mass spectrometryanalysis, there are several limitations. Phosphopep-tides could be lost due to their inability to bind to theIMAC column. In addition, multiple phosphorylatedpeptides are more enriched and may have difficultyin elution. A major limitation may also be the non-specific binding of nonphosphorylated peptides suchas acidic peptides (rich in glutamic and asparticacid). The conversion of peptides to their corre-sponding methyl esters proposed by Ficarro et al. notonly significantly increased IMAC selectivity forphosphopeptides but also illustrated the potential ofIMAC-based enrichment for global phosphopro-teome analysis.(15)

Another highly specific phosphopeptide isola-tion was demonstrated by Pinkse et al. using on-linetitanium dioxide (TiO2) columns.(16) The approachwas based on the selective interaction of phosphateswith porous titanium dioxide microsphere via biden-

Fig. 1 Overview of protein phosphorylation analysis usingmass spectrometry.

phosphopeptideenrichment

IMAC, TiO2,chemical modification recognition of

phosphopeptidesphosphatase treatment

neutral loss scanprecursor ion scan

proteolysis

phosphoproteinenrichment

Immunoprecipitation

Proteins

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tate binding at the TiO2 surface.(17) Larsen et al. sub-sequently modified the method by packing the TiO2

beads in GELoader tips as TiO2 microcolumns andpeptide loading in 2,5-dihydroxybenzoic acid(DHB), which showed less non-specific binding thanIMAC.(18) Titanium dioxide has been successfullyused for mapping phosphorylation sites at low fem-tomole levels in combination with multiple proteincleavage using different protease and sensitivenanoLC-MS/MS.(19) In addition, the use of magneticFe3O4/TiO2 core/shell nanoparticles as affinity probesto concentrate phosphopeptides has been reported.(20)

Nevertheless, few phosphoproteome analyses usingTiO2 enrichment have been reported so far, and theiraccessibility for large scale protein phosphorylationanalysis still need to be investigated.

Aside from IMAC and TiO2 enrichment, severalmaterials have also been mentioned to be able toenrich phosphopeptides/phosphoproteins. The use ofa metal hydroxide, Al(OH)3, was proved effectiveand more selective than commercial phosphoproteinenrichment kits.(21) The utility of zirconium dioxide(ZrO2) microtips for phosphopeptide isolation priorto mass spectrometric analysis has been demonstrat-ed displaying similar overall performance as TiO2

microtips.

Chemical modification methods

Chemical replacements of the phosphate groupby an affinity group or tags that can be recognizedusing mass spectrometry analysis are alternativestrategies of phosphopeptide/phosphoprotein enrich-ment. Both phosphoserine- and phosphothreonine

containing peptides can lose H3PO4 by β-eliminationreaction under high pH, resulting in dehydroanalineand dehydroaminobutyric acid residues.Ethanedithiol (EDT), acts as a nucleophile, reacts todouble band, and provides a new reactive thiolgroup. Subsequent reaction of the free thiol groupwith different biotin derivatives enables the isolationof labeled peptides by an avidin chromatography(Fig. 2A).(22) To avoid side reactions, the thiol groupson the cysteine must be blocked prior to the reaction.Goshe et al. proposed a modified strategy coupledwith labeled EDT (H3 or D4-ethanedithiol), calledphosphoprotein isotope-coded affinity tags (PhIAT),for not only the isolation but also the quantitation ofphosphopeptides.(23) Goshe et al. even expanded itsapplication for enrichment and identification of lowabundance phosphoproteins by integrating capillaryreversed-phase liquid chromatography for separatingthe recovered peptides.(24) These chemical modifica-tion strategies encounter a main constraint that theyare not suitable for tyrosine phosphorylation sincetyrosine phosphorylated residues seldom undergo β-elimination. However, this procedure is easy to per-form and reduced the losses due to the complicatedreactions.

Another chemical modification method, report-ed by Zhou et al., was comprehensively applicable toall phosphopeptides (Fig. 2B).(25) Using a carbodi-imide condensation reaction, the cystamine groupwas attached to the phosphate moiety. Purification ofthe modified peptides was accomplished by attach-ment to aodoacetyl resin and released by treatmentwith trifluoroacetic acid. To avoid unwanted reac-

Fig. 2 Schemes for enrichment approach using chemical modification. (A) Oda et al.(22) utilized ethanedithiol to provide a thiolgroup, allowing subsequent reaction with a affinity biotin tag. (B) Zhou et al.(25) couple thiol tags to phosphate groups by a carbodi-imide activation. Tagged peptides are bound to iodoacetamide-functionalized beads, released by TFA.

A

B



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tion, the amino groups and the carboxyl groups ofthe peptides were blocked with tert-butyl oxycar-bonyl (tBoc) chemistry and amidation, respectively.This approach required multiple chemical reactionand purification steps before mass spectrometryanalysis, which could introduce great losses.

Phosphoproteins can be enriched by specificanti-phospho antibodies, which greatly reduce thecomplexity of proteome. Anti-phosphotyrosine anti-bodies are currently available that can efficientlyimmunoprecipitate tyrosine phosphorylated proteins.However, anti-phosphoserine or anti-phosphothreo-nine antibody does not give the similar efficiency,which confines the studies of the complete phospho-proteome using this method. In addition, theenriched proteins are still too complicated for phos-phorylation analysis after enzymatic digestion. It canbe ameliorated by coupling with a chromatographicenrichment. IMAC or TiO2 has become a commontool for phosphopeptide enrichment. No experimen-tal evidence has shown inconsistencies in bindingability between serine-, treonine-, and tyrosine-phos-phorylated species to those chromatographicapproaches, which can be used for large-scale andcomprehensive phosphoproteome analysis. Thegreatest limitation of the technique has been the non-specific binding of acidic peptides. Even though sev-eral chemical modifications enrichment strategieshave been proposed to enhance the selectivity, theyrequire multiple steps for chemical reactions and thereaction conditions must be well-controlled to getbetter yields. The existing methods require furtherimprovements or refinements and should be com-bined to augment purity of phosphopeptides.

Recognition of phosphopeptidesPhosphatase treatment

Phosphatase can remove phosphate group(s)from phosphopeptides, yielding a “mass shift” rela-tive to the original mass of the phosphopeptide.Depending on the number of phosphate groups, themass shift could be -80 Da (HPO3 = 79.966) or itsmultiples (–80 n Da, where n is the number ofphosphate groups). Phosphopeptide identification orsequencing can be achieved during further experi-ments by focusing on the signals that exhibit massshifts after phosphatase treatment. Liao et al. appliedthe combination of phosphatase treatment and MS-based identification. This approach was applied to

analyze phosphopeptides using matrix-assisted laserdesorption/ionization time-of-flight mass spectrome-try (MALDI-TOF-MS) before and after being treatedwith a phosphatase to differentiate phosphopeptidesignals from others (Fig. 3).(26) Several researchersalso have demonstrated that phosphopeptidesenriched by IMAC or fractionated using reverse-phase High performance liquid chromatography(HPLC) can be identified by observing the mass shiftin the MALDI spectrum following phosphatase treat-ment.(27-31)

In 2005, Torres et al. developed a strategynamed Phosphatase-directed Phosphorylation-siteDetermination (PPD).(32) Because dephosphorylatedpeptides are known to be more detectable in the MS,IMAC-enriched peptides were first treated withphosphatase to yield dephosphorylated peptide sig-nals. Their sequences were consequently defined byMS/MS analysis and the total number of Ser, Thr, orTyr residues could hypothetically be used to predictthe location of phosphopeptide signals. On the basisof this information, a mass list was used to directMALDI-MS/MS on the phosphorylated peptidesbound to IMAC beads for phsphorylation site deter-mination.(32) Although this approach was powerful forphosphoprotein analysis, it is not applicable for com-prehensive analysis.

During high-throughput phosphopeptide identi-fication, database search tools may categorize largenumbers of false-positive/false-negative phospho-peptide assignments due to their inefficient fragmen-tation. A scheme containing a dephosphorylationreaction was used to increase the reliability of phos-phopeptide identification results in a comprehensivestudy by choosing the peptide sequencing resultswith mass shifts and close retention times before andafter phosphatase treatment.(33)

Recently, we proposed a strategy to mine phos-phopeptide signals by observing mass shifts generat-ed from dephosphorylation reaction in liquid chro-matography-mass spectrometry data (Fig. 4).(34) LC-MS analysis was performed on TiO2-enriched pep-tides before and after phosphatase treatment. Realphosphopeptide signals were expected to emergemass shifts between the two LC-MS data set due toloss of phosphate moiety. Since LC-MS analysis maygenerate tremendous amount of signals, manualinterpretation of these spectra may be very tediousand inefficient. We programmed an in-house com-



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puter program, DeltaFinder, which is used to processdata and differentiate possible phosphopeptide sig-nals. The potential signals were sorted out of thecomplicated LC-MS data set. As shown in Fig. 5A,peptide mixtures of α- and β-caseins revealed that 61peptide signals consisting of phosphopeptides alongwith many nonphosphorylated peptides wereobserved after TiO2 enrichment. Followed by analkaline phosphatase treatment, 93 peptide masseswere present in the LC-MS data (Fig. 5B). Amongthem, only 10 pairs were referred to as potentialphosphopeptides and their counterparts after beingprocessed by the DeltaFinder (Fig. 5C). The reten-tion times and m/z values of these selected LC-MSsignals were used to facilitate subsequent LC-MS/MS experiments for phosphorylation site deter-mination. This scheme has showed its capability ofidentifying more phosphorylation sites, in compari-son with conventional data-dependent LC-MS/MSexperiments, in the mixture of α- and β-caseins. Foranalyzing phosphoproteome, our approach also dom-inated over conventionally-used mass spectrometricanalysis sets in the data-dependent mode and defined

much more phosphopeptides as well as sites. Theresults shown in this work also demonstrated thevalue of computational algorithms for not only pro-cessing the data but also assisting in phosphorylationanalysis.

Mass-spectrometric detection

Except for the examination of mass shifts, thespecific ions derived from the side chains of phos-phorylated residues, which are called “reporter ions”,can also be used to confirm the existence of phos-phorylation. Therefore, several mass spectrometrictechniques have been employed to distinguish thoseions.

Precursor ion scanPhosphate groups carried by peptides have the

tendency to lose ions during fragmentation underhigh pH conditions and give rise to phosphate-derived anions at m/z 79 (PO3

-) as a reporter ion. Amass spectrometer operating in the negative ionmode is set to detect this particular signal in the pre-cursor ion scans. In contrast to the neutral loss scans,

Fig. 3 The mass shift from a phosphopeptie signal can be observed in MS analysis after dephosphorylation of three standard phos-phopeptides. Figure adapted from reference 26.

Rel

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e In

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Rel

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1424KRPpSQR-HGSKY-amide

910KRpTLRR

900LKRApYLG-amide

Dephosphorylation

820LKRAYLG-amide

–80

900

Dephosphorylation

830KRTLRR

–80910

Dephosphorylation

1424

–80

1344

KRPSQR-HGSKY-amide

1200 1300 1400 1500 1600m/z

700 800 900 1000 1100m/z

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phosphoserine-, phosphothreonine, and phosphotyro-sine-containing peptides can yield this reporter ion inthis method, which has been proved as a sensitivemethod for analyzing phosphopeptides.(35,36) The limi-tation of this method is that the sample must be acid-ified before being analyzed in the positive mode forpeptide sequencing. Precursor ion scanning for theimmonium ion of phosphotyrosine at m/z 216 can beperformed in the positive ion mode, allowing for thesubsequent sequencing of the corresponding phos-phopeptides.(37,38)

Neutral loss scanIn positive ion tandem MS, phosphoserine- and

phosphothreonine-containing peptides prefer toundergo β-elimination reaction and give rise to aneutral loss of 98 Da (H3PO4) or 80 Da (HPO3). Thisphenomenon can be used for the selective detectionof phosphopeptides.(39-41) In the MS/MS spectra, theloss of phosphoric acid (H3PO4) converts phospho-serine and phosphothreonine residues into dehy-droalanine (69 Da) and dehydroaminobutyricresidues (83 Da) respectively, pointing out the exactlocation of phosphoserine and phosphothreonineresidues. In contrast, relatively few phosphoric acidneutral fragments were observed for phosphotyro-sine-containing peptides. The phosphotyrosine-con-taining peptides are typically stable under these con-

A program exports list of phosphorylated anddephosphorylated peptide signal pairs (Mi and Mj)

Mi = Mj + (80 x n), n = 1, 2, 3...

p

pp

LC-MS

Mi Mj

y b

b

LC-MS

LC-MS/MS of Mi

Add Alkaline Phosphatase

Phosphatemoiety

Peptide Mixture

TiO2 Microcolumn Enrichment

Non-binding Peptides

Binding Peptides

Fig. 4 A newly proposed strategy for analysis of protein phosphorylation by taking advantage of detecting mass shift derived fromdephosphorylatrion of phosphopeptides. The succeeding LC-MS/MS analysis of those selected potential phosphopeptide signalsreveal the identification of phosphopeptides. Figure adapted from reference 34.

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ditions, which hampers the applicability of thismethod for tyrosine phosphorylation analysis. Takethe work of Gruhler et al. as an example, usingMS/MS and neutral loss-dependent MS3, Only fourof 729 phosphorylation sites (0.5%) were identifiedas tyrosine phosphorylation in a yeast phosphopro-teome.(42) Therefore, neutral loss scanning appears toattenuate for the detection of tyrosine phosphoryla-tion.

Fluorescent affinity tags

A novel fluorescent affinity tag (FAT) was syn-thesized and used to selectively modify phosphory-lated serine and threonine residues by beta-elimina-

tion and Michael addition. Fluorescence imagingwas performed after a solution- or gel-based separa-tion method to visualize these tagged phosphopep-tides. The strong fluorescence signal enhanced thedetection of phosphoproteins, allowing the subse-quent identification.(43)

Precursor ion scanning for ions producing a 79Da fragment is useful because of the sensitive MSdetection of the PO3

- anion. The pitfall is the require-ment to work in the negative mode, making the useof the data in conjunction with on-line HPLC diffi-cult. Although phosphotyrosine immonium ion isdetected at 216, immonium ions for phosphoserinesand phosphothreonines are rarely seen because they

Fig. 5 Three dimensional plots of LC-MS data obtained from (A) the tryptic digest of α-and β-casein followed by TiO2 microcol-umn enrichment, and (B) its additional treatment with alkaline phosphatase. In (C), only 10 probable phosphopeptide signals pickedby the program are shown. Figure was modified from reference 34.

A B

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are prone to lose phosphate groups. On the contrary,scanning for neutral loss of H3PO4 can hardly detectphosphotyrosine since it is apt to lose HPO3 (80 Da)rather than H3PO4. Recognizing phosphopeptides byobserving mass shifts owing to treatment with phos-phatase may introduce less or no selection bias sincethe dephosphorylation reaction is universal to ser-ine-, threonine-, and tyrosine-phosphorylated pep-tides/proteins.

Quantification of phosphorylationThe conventional methods used for analyzing

stoichiometry of protein phosphorylation are phos-phoamino acid analysis or Edman degradation after32P incorporation.(44,45) It is a tedious procedure andrequires handling large amounts of radioactive sub-stances.

Oda et al. used stable isotopes to quantify phos-phorylation events by growing two different cellpopulations in the presence of 15N-labeled or 14N-labeled medium.(46) In addition, stable isotope label-ing using amino acids in a cell culture (SILAC) wasperformed by growing cells with different isotopical-ly labeled amino acid such as [13C6]arginine and[13C6]lysine. Using SILAC in combination with LC-MS/MS, Gruhler et al. characterize phosphorylationsites in yeast that are regulated during the matingresponse.(42)

A strategy using phosphoprotein-specific iso-tope-coded affinity tags (PhIAT) has been pro-posed.(23) PhIAT are biotin-containing affinity tagsthat can be introduced to phosphopeptides by way ofthe β-elimination reaction.(23) It enables both purifica-tion and quantitation of phosphoserine- and phospho-threonine-containing peptides. Another approach,proposed by Weckwerth et al., incorporates H5- orD5-ethanethiol as a nucleophile. However in theirstudy, no further biotin addition and thus no enrich-ment was performed.(47)

A stable isotope-tagged amine-reactive reagent,isobaric tag for relative and absolute quantitation(iTRAQ), were design to be isobaric during MS andfragment during MS/MS to reveal differential lowmass ions. It consisted of a reporter group (mass =114-117), a balance group (mass = 31-28), and a pep-tide reactive group. Four iTRAQ reagents allowedfour samples to be compared in a single analysis.Combining labeled peptides from different treat-ments, relative quantification were accomplished by

inspecting the relative responses of reporter groupsgenerated from neutral loss during MS/MS experi-ments. Zhang et al used iTRAQ to successfully iden-tify the dynamics of tyrosine phosphorylation inresponse to epidermal growth factor (EGF) in epithe-lial cells.(48)

Phosphoproteome analysisIt has been estimated that the human genome

contains 518 genes for kinases, thus, is one of thelargest protein families. In addition, 30% of all cellu-lar proteins may be phosphorylated at any time, indi-cating that the phosphoproteome of each organism isvast and plays a vital role in regulating cellular activ-ities.(49) There are approximately 100000 potentialphosphorylation sites in the human proteome ofwhich fewer than 2000 are currently known.Knowing the phosphoproteome would be a valuableasset in understanding phosphorylation-based signal-ing networks. The existing mass spectrometry-basedmethods make the investigation of the phosphopro-teome on a global scale possible. We have foundmany cases in which phosphoproteome analyseshave been described. According to those investiga-tions, enrichment of phosphorylated species beforeMS plays an important role in introducing successesof the characterization of phosphorylation in intricatesamples. Among them, IMAC demonstrated muchmore success than TiO2 microcolumns, Furthermore,the application of TiO2 enrichment for phosphopro-teome-wide scale analysis requires more investiga-tion.

ConclusionsDespite the fact a number of analytical strate-

gies have been developed for the characterization ofprotein phosphorylation, there is no single methodthat is superior to others for the identification of pro-tein phosphorylation sites. No matter what methodsare used, enrichment of phosphorylated proteins andpeptides increase the probability of success. With theincreasing number of improvements of the massspectrometry and sample preparation techniques, wecan envisage the accessibility of a comprehensiveapproach for the analysis of protein phosphorylation.

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Analysis of protein phosphorylation by mass spectrometry

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