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Using isotopically-coded hydrogen peroxide as a

surface modification reagent for the structuralcharacterization of prion protein aggregates☆

Jason J. Serpaa, Karl A.T. Makepeacea, Tristan H. Borchersa, David S. Wishartb,Evgeniy V. Petrotchenkoa, Christoph H. Borchersa,c,⁎aUniversity of Victoria, Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park,Victoria, BC V8Z7X8, CanadabDepartments of Biological Sciences and Computing Science, University of Alberta, Edmonton, Alberta, T6G 2E8, CanadacUniversity of Victoria, Department of Biochemistry &Microbiology, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada

A R T I C L E I N F O

☆ This article is part of a Special Issue entit⁎ Corresponding author at:University of Victor

Technology Park, Victoria, BC, V8Z7X8, CanaE-mail address: christoph@proteincentre.

1874-3919/$ – see front matter © 2013 Elseviehttp://dx.doi.org/10.1016/j.jprot.2013.11.020

A B S T R A C T

Available online 3 December 2013

The conversion of the cellular prion protein (PrPC) into aggregatedß-oligomeric (PrPß) and fibril(PrPSc) forms is the central element in the development of prion diseases. Here we report thefirst use of isotopically-coded hydrogen peroxide surface modification combined with massspectrometry (MS) for the differential characterization of PrPC and PrPβ. 16O and 18O hydrogenperoxide were used to oxidize methionine and tryptophan residues in PrPC and PrPβ, allowingfor the relative quantitation of the extent of modification of each form of the prion protein.Aftermodificationwith either light or heavy forms of hydrogen peroxide (H2

16O2 andH218O2), the

PrPC and PrPβ forms of the proteinwere then combined, digestedwith trypsin, and analysed byLC-MS. The 18O/16O signal intensity ratios were used to determine the relative levels ofoxidation of specific amino acids in the PrPC and PrPβ forms. Using this approach we havedetected several residues that are differentially-oxidized between the native and β-oligomericprion forms, allowing determination of the regions of PrPC involved in the formation of PrPβ

aggregates. Modification of these residues in the β-oligomeric form is compatible with a flip ofthe β1-H1-β2 loop away from amphipathic helices 2 and 3 during conversion.

Biological significanceSurface modification using isotopically-coded hydrogen peroxide has allowed quantita-tive comparison of the exposure of methionine and tryptophan residues in PrPC and PrPß

forms of prion protein. Detected changes in surface exposure of a number of residues haveindicated portions of the PrP structure which undergo conformational transition uponconversion.This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomicsand Phenotypes?

© 2013 Elsevier B.V. All rights reserved.

Keywords:Mass spectrometryOxidative labelingPrion aggregate structureChemical surface modificationStructural proteomicsStable isotope labeled hydrogenperoxide

led: Can Proteomics Fill the Gap Between Genomics and Phenotypes?ia, GenomeBritish Columbia Proteomics Centre, #3101-4464MarkhamStreet, Vancouver Islandda. Tel.: +1 250 483 3221; fax: +1 250 483 3238.com (C.H. Borchers).

r B.V. All rights reserved.

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1. Introduction

The conversion of the cellular prion protein (PrPC) into theaggregated ß-oligomeric (PrPß) and fibril (PrPSc) forms is thecentral element in the development of prion diseases [1]. Thistransition results in the formation of the insoluble form of theprotein, which leads to an accumulation of amyloid fibrils inthe central nervous system and eventually to death. Neitherthe final structure of the aggregates, nor the exact molecularmechanisms that lead to the aggregation are known. Thesecomplexes are difficult to characterize by traditional methodsfor studying protein structure, such as X-ray crystallographyand conventional NMR spectroscopy, because of problemswith crystallization and solubility. Therefore, the study ofprion conformational change is an ideal case for the use ofalternative approaches, including structural proteomics.

Structural proteomics can be defined as the combination ofprotein chemistry methods with mass spectrometry. Thesemethods include limited proteolysis, surface chemical modifica-tion, hydrogen/deuterium exchange, and chemical crosslinking.When combined with contemporary mass spectrometry, thesemethods can provide detailed structural information for proteinsand protein complexes [2,3].

Chemical surface modification of the protein surfaceallows the determination of the regions of the proteinswhich are exposed to the solvent, whichmakes this techniqueparticularly suitable for the characterization of the conforma-tional changes in proteins. Identification of the actualmodification sites indicates which specific amino acid resi-dues are on the protein surfaces and therefore in contact withthe solvent. Determining the differences in reactivity ofamino acid residues between different conformational statesof the protein enables localization of regions of proteinsurfaces which undergo transition during these conforma-tional changes. Recently, mass spectrometry has become themethod of choice for determining the extent of modificationof specific amino acid residues in proteins, and the use of theisotopically-coded modification reagents allows the quantita-tion of the modification yield by mass spectrometry. UsingMS, distinct stable isotopic forms of the reagents, which differin mass, but are otherwise chemically identical, can be usedfor the comparison of the separate samples. Combining thedifferentially modified reaction samples allows detection ofthe products within the same mass spectrum, thus eliminat-ing run-to-run signal variability and providing reliable relativequantitation of the reaction products. Several examples ofapplying such isotopically-coded chemical surface modifica-tion reagents to the study of protein structure with massspectrometry have recently been reported [2,4].

Oxidation of amino acid residues can be considered as aparticular type of surface modification, and different types ofoxidation reactions can be used for this purpose. Thesemethods include the use of hydroxyl radicals (·OH) as a covalentlabeling agent [5–7] and FPOP (fast photochemical oxidation ofproteins) [8,9]. Taking advantage of the availability ofisotopically-coded hydrogen peroxide, we decided to combineoxidative labeling with isotopic coding of the H2O2 reagent inorder to achieve facile relative quantitation of the oxidativereaction between two different conformational states of the

prion proteins. H2O2 has been successfully used to analyseprotein-ligand binding of multi-component protein mixtures[10]. H2O2 can beused to specifically oxidizemethionine residues[11,12] and tryptophan [13,14]. In vitro methionine oxidationstudies of PrPC using hydrogen peroxide have also beenperformed [12,15–19]. In these experiments, we used the sameexperimental conditions for oxidation that were used previouslyby Requena et al. [12], where the results were verified by CD. Webelieve that under these experimental conditions, we mainlycapture the initial stages of these modification reactions.

In the case of prion protein, where there are at least twoconformationally different states, a comparison of the oxidationlevels of specific methionine and tryptophan residues betweenthese states can provide details of their structural differences.However, obtaining an accurate quantitative comparison ofamino acid residues oxidation reactivities for different prionforms would be difficult without using isotopically-labeledreagents. To quantify the oxidation levels between two differentconformational states of PrP, we therefore used light and heavyisotopic forms of the H2O2 reagent. Using this approach, we candirectly compare the relative reactivity of specific amino acidresidues between the PrPC and PrPß forms. This, in turn, allowedus to determine changes in the surface exposure of specificmethionine and tryptophan residues, and, from these changes,we were able to determine the structural differences betweenthese two prion forms.

2. Materials and methods

All chemicals were from Sigma–Aldrich, unless noted other-wise. Native and β-oligomeric Syrian hamster 90-232 prionproteins [20] were obtained from PrioNet's PrP5 facility(University of Alberta, Canada).

The model peptide (-)EGFRCHMLPSPTDSNFYR was used tooptimize the reaction conditions. PrPC and PrPβ samples werebubbled with nitrogen gas (Supplementary Fig. 1) prior tomodification with 10 mM (final concentration) heavy or lightH2O2. Reactions were performed in triplicate (three replicateswith PrPC + H2

16O2 and PrPß + H218O2, and three replicates with

PrPBß + H216O2 and PrPC + H2

18O2), at 37 °C for 15 min (Fig. 1).Modified PrPC and PrPβ samples were quenched with 150 mMmethionine (Supplementary Fig. 2) and combined. Samplesweremixed with equal volumes of 8 M urea, bubbled with nitrogengas, and incubated at 20 °C for 2.5 h. DTT was added to give10 mM final concentration, and incubated at 37 °C for 30 min.Samples were then diluted five-fold with PBS pH 7.4 anddigested with trypsin at a 1:1 (w/w) ratio at 37 °C for 6 h.Completeness of digestion for all samples was confirmed bySDS-PAGE. Samples were further reduced by the addition ofTCEP to give a final concentration of 5 mM, and acidified withTFA to give a 0.1% final concentration, prior to LC-ESI-MS andMS/MS analysis.

Mass spectrometric analysis was performed with a nano-HPLC system (Easy-nLC II, ThermoFisher Scientific, Bremen,Germany) coupled to the electrospray ionization source of anLTQ Orbitrap Velos mass spectrometer (ThermoFisher Scientif-ic). Samples were injected onto a 100 μm ID, 360 μm OD trapcolumn packed with Magic C18AQ (Bruker-Michrom, Auburn,CA), 100 Å, 5 μm pore size (prepared in-house), and desalted by

Fig. 1 – Experimental scheme for differential modification of the native and β-oligomeric forms of the prion protein with H216O2

and H218O2. The isotopic distributions shown are for oxidized peptides from PrPC (green) and PrPβ (blue), with intensity ratios of

2:1. These distributions were derived from PrPC + H216O2 and PrPβ + H2

18O2 (left), and from the alternative labeling scheme withPrPC + H2

18O2 and PrPβ + H216O2 (right).

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washing for 15 minwith 0.1% formic acid (FA). Separations weredone on a 75 μm ID, 360 μm OD analytical column packed(in-house) withMagic C18AQ, 100 Å particle size, 5 μmpore size,and with an IntegraFrit (New Objective Inc. Woburn, MA). Thecolumnwas equilibratedwith 95% solvent A (2% acetonitrile and98% water, both containing 0.1% FA) before the peptides wereseparated with a 60 min acetonitrile:water gradient. The gradi-ent used was 0–60 min: 4–40% B, 60–62 min: 40–80% B, 62–70 min: 80%B, with solvent B containing 90% acetonitrile and10% water, both 0.1% FA.

MSdatawere acquired indata-dependentMS/MSmodewiththe six most intense peaks in each full MS scan selected forfragmentation. Dynamic exclusion was set to 60 s with repeatcount of 2. MS scans (m/z 400–2000 range) and MS/MS scanswere acquired at 60,000 and 30,000 resolution, respectively. MS/MS fragmentation was performed by collision-induced dissoci-ation activation at a normalized collision energy of 35% andmeasured in the FT.

Proteome Discoverer (Ver. 1.4.0.288) was used to generate.MGF files from.RAW files. Oxidized peptideswere identifiedwithMASCOT using a custom Syrian hamster prion sequencedatabase, with open enzyme cleavage, oxidation of methionine,tryptophan, histidine, and tyrosine residues as variable modifi-cations, peptide mass tolerance 8 ppm, MS/MS mass tolerance0.03 amu, and instrument set at ESI-TRAP. The list of oxidizedpeptides identified by MASCOT was compiled for all runs,grouping those peptideswith similar retention times and highestMASCOT ion scores. Each modified peptide assignment wasverified manually.

For each modified peptide containing one modificationfrom each PrPC + H2

16O2/PrPBß + H218O2 and PrPBß + H2

16O2/PrPC +H218O2 run we used Thermo Xcalibur 2.2 Qual Browser to obtain

the MS isotopic cluster. Isotopic distributions of the oxidizedpeptides were obtained using the MS-Isotope program inProtein Prospector [21]. From these distributions, a ratio of the3rd isotopic peak versus the 1st isotopic peak (rnat) was

calculated. The experimental ratios of the 3rd isotopic peakversus the 1st isotopic peak of the labeled and reverse-labeledoxidized peptides (rβC and rCβ) were also calculated. Differ-ences in oxidation levels between PrPC and PrPß samplesweredetermined by comparing 18O oxidation contributions usingFormula 1. Residues with a higher oxidation level in the PrPC

form have values less than 1.0, while residues with higheroxidation levels in PrPß have values greater than 1.0.

rnat−rβc−1� �

rcβ−rnat� �

rnat−rcβ−1� �

rβc−rnat� � Formula1

3. Results and discussion

Oxidation of amino acid residues, in particular oxidation ofmethionine, can occur during the general handling of aprotein [22–24], during the electrospray ionization process[25,26] and/or during SDS-PAGE [27,28]. With this approach,the background oxidation is superimposed on the oxidationlevels created by the modification reactions. We balance outthe effects of the background oxidation by additional exper-iments with reversal of isotopic forms of H2O2 for themodification, so that the contribution of background oxida-tion is negated.

The calculation of differential oxidation levels could be donein a number of different ways. In the study of protein–ligandbinding using labeled hydrogen peroxide [10], DeArmond et al.quantified extent of oxidation using weighted averagemolecularweight representative of eachoxidizedpeptide [10].Wehave triedthis method calculation on a number of PrPC and PrPß oxidizedresidues, and found results that were comparable with thoseobtained using the comparative ratio formula presented here.

It has been reported that the methionines of PrP areparticularly prone to oxidation [12,19,29], and it has been

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shown that conditions employed here can be used for PrPstudies as the reactions are relatively rapid. It has also beenshown that similar reaction conditions result in only minimalconformational change of the prion [12]. Furthermore, the useof H2O2 without metal results in only one oxidation ofmethionine to methionine sulfoxide and not the secondoxidation to methionine sulfone [12].

Although it may be tempting to pursue oxidation of otheramino acid residues in order to obtain more detailed structuralinformation, the use of harsher or alternative oxidation tech-niques, such as combination of hydrogen peroxide with metalions, at least in the case of prion, can alter the conformation ofthe prion form being studied. PrP is a protein with high affinity,histidine-based copper binding sites [30,31]. As such, it is verysusceptible to metal-catalyzed oxidation. There are a number ofadditional amino acid residues (tyrosine, histidine) which wewould expect to be oxidized under these conditions, but thereaction will also result in aggregation of PrP resulting fromhistidine oxidation [17] and has been shown to initiate aggrega-tion of the protein [17] and PK resistance [32]. Furthermore, theaddition of H2O2 and Cu2+ leads to widespread fragmentation ofPrP [33].

Using the approach of isotopically-coded H2O2 modification(Fig. 1), we detected several residues that were differentiallymodified between the PrPC and PrPβ prion isoforms. Differentialmodification levels were calculated by superimposing the MSisotopic distributions of aH2

18O2modified peptide and the known

Fig. 2 – Mass spectra of oxidized peptides containing differentially mthePrPC andPrPβ formsarehighlightedwith greenandblueboxes, rebeenmodified with H2

18O2. Bottom: PrPC has been modified with H218

modification levels for two forms of the protein can be calculated byH216O2- and H2

18O2-modified peptides.

isotopic distribution of H216O2 modified peptide, leading to a ratio

representing the difference in modification between two formsof the prion protein (Fig. 2). PrPC and PrPβ were alternativelylabelled with H2

16O2 and H218O2 to account for any possible

differences in reactivities between the H216O2 and H2

18O2 prepara-tions, and any possible background oxidation of the proteinsamples prior to MS analysis. The analyses were performed intriplicate and detected nine of the ten methionines and one ofthe two tryptophan residues (Table 1). We observed differentialmodification of multiple residues in both PrPC and PrPβ forms ofprion (Fig. 3), with many of the methionine residues localized inwhat are already known to be critical regions for the conforma-tional changeof theprionprotein [12]. Differences inoxidationofthese residues can give insights into structural changes thatoccur during PrPC to PrPβ conversion.

A comparative ratio of 1.01 was found for M87 indicative ofa residue with similar oxidation levels in both PrPC and PrPβ.M87 is a part of an artificially introduced N-terminal tag and islocated close to the beginning of the flexible N-terminalregion. An equal modification rate of this residue most likelyindicates its equal accessibility, due to the absence of adefined structure at the very N-terminus in both forms. Thiscan serve as an internal control of the labelling efficiency forboth the light and heavy forms of the modification reagents,and as a confirmation of the ratio calculations.

Several residues were preferentially modified in the PrPC

form: M129, M138, and M213. M129 is located in the beginning

odified residues M138 andM206. Contributions of signals fromspectively. Top: PrPChasbeenmodifiedwithH2

16O2 andPrPβhasO2 and PrPβ has beenmodified with H2

16O2. Differences indeconvoluting the superimposed isotopic distributions from the

Table 1 – Differentially oxidized amino acids of PrPC and PrPβ. The ratios (rcβ and rβc) representing the differentialmodification of the specific residues are calculated as an average of three pairs of replicate runs, which are then input intoFormula 1 from which the comparative ratio is calculated. Values greater than 1.0 indicate preferential modification of theresidues in the PrPß form, while ratios less than 1.0 indicate preferential modification of the residues in the PrPC form.

Residue Sequence rcβ rβc Comparative ratio PrPC PrPβ

Average SD Average SD

M87 (R)GSHMLEGQGGGTHNQW(N) 0.70 0.09 0.69 0.10 1.01 X XW99 (R)GSHMLEGQGGGTHNQW(N) 0.44 0.04 0.47 0.04 1.96 XM112 (K)HMAGAAAAGAVVGGLGGY(M) 0.61 0.17 0.73 0.08 1.76 XM129 (A)GAVVGGLGGYMLGSAMSR(P) 0.88 0.26 0.92 0.04 0.92 XM134 (A)GAVVGGLGGYMLGSAMSR(P) 0.78 0.11 0.68 0.21 1.59 XM138 (R)PMMHFGNDWEDR(Y) 0.66 0.09 0.74 0.21 0.72 XM139 (R)PMMHFGNDWEDR(Y) 0.60 0.19 0.57 0.15 1.31 XM206 (K)GENFTETDIKIMER(V) 1.45 0.59 0.65 a 0.02 3.85 XM213 (R)VVEQMCTTQYQK(E) 0.41 0.05 0.60 0.07 −0.19 X

a Note: this peptide was found in only two of the three runs.

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of the β-sheet 1 of PrPC. This region is thought to be involvedin the formation of the final stacked β-sheet structure in theamyloid fibrils and has been previously shown to be stronglyprotected from the solvent in this form of PrP [34]. Based onour data, there is only mild preferential protection of thisresidue, whichmay imply that an extensive β-sheet structureis not formed yet in the β-oligomer form. This is in agreementwith our previous findings on the exposure of tyrosineresidues, based on modifications with pyridine carboxylicacid N-hydroxysulfosuccinimide ester (PCASS), in this regionof the PrPβ [2]. The M138 reside is located within the strand,between β1 and H1 strand, and is readily exposed in the PrPC.Rearrangement of the β1-H1-β2 loopmight therefore accountfor a change in the microenvironment of this residue in PrPβ.M213 is the part of the same region – the β1-H1-β2/H3interface – and is also situated on the protein surface in the

Fig. 3 – Oxidized residues highlighted on the PrPC structure.Residues that are preferentially oxidized in PrPC and PrPβ aremarked in blue and red, respectively; residues that are equallyoxidized are marked in purple; residues, for which singlyoxidized peptides were not detected are marked in grey.

PrPC. Moving the β1-H1-β2 loop away from the H2-H3 corewould open up the hydrophobic internal region of β1-H1-β2/H2-H3, where M213 is located. Our crosslinking data on thePrPβ (manuscript in preparation) suggest that this regionbecomes occupied with another portion of the prion mole-cule in the β-oligomer aggregate. This would therefore be inagreement with the absence of the oxidation of this residuein PrPβ, which we observed in this study.

A number of residues were preferentially modified in thePrPβ form: W99, M112, M134, M139, and M206. The W99 andM112 residues are located in the N-terminal flexible portion ofthe 90-232 PrP molecule. We proposed recently that this regionmay transiently interact with the PrPC protein surface which iscomposed of the C-terminal portion of H2 and the H2-H3 loop[35]. The current data may suggest relocation of this region inPrPβ. M134 and M139 are located in the β1-H1 region with sidechains oriented towards the interior H2-H3 core in PrPC

structure. Rearrangement and movement of the β1-H1-β2 loopaway from the PrP core would then expose this region to thesolvent if it is doesnot get involved in any additional contacts inPrPβ.

Overall, these current results on the H2O2 differentialoxidation of PrPC and PrPβ are in good agreement with ourprevious surface modification results, performed using reagentPCASS [2]whichpreferablymodifies lysines and theN-terminusbut also reacts with tyrosines, serines, and threonines. WithPCASS, Y150 andY157 showedpreferentialmodification in PrPβ,which shares the same internal face of the β1-H1-β2 loop withM134 and M139 [2]. The same movement of H1 away from thecorewould also allow H2O2 access to the M206 residue, which issituated internally between C-terminal part of H2 andN-terminal part of H3. Furthermore, these oxidation resultsare in good agreement with our hydrogen/deuterium exchangeexperiments on PrP indicating rearrangement of the sameregion [2]. Specifically, previous HDX results indicated residues148-164 (H1-β2) [2] and 132-167 (β1-H1-β2) [36] as beingdeprotected in the oligomeric form. As evidenced with ourPCASS surface modification studies [2], the rearrangement ofthe β1-H1-β2 in the oligomeric form can lead to an increasedexposure of residues sharing the interface of the β1-H1-β2 loop

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with the H2-H3 core and, in the case of this study, results inincreased exposure of residues M134, M139, and M206.

4. Conclusions

Wehave presented here the use of 16O and 18O isotopically-codedhydrogen peroxide oxidation as a surface modification reagentfor the relative quantitation of the extent of modification ofmethionine and tryptophan residues between two conforma-tional states of a protein. This approach allowed the detection ofseveral differentially oxidized residues between the native andβ-oligomeric forms of the prion protein. The differences foundcanbeexplainedbya flip of theβ1-H1-β2 loopaway fromthe coreof the molecule during conversion and indicate specific residuesof prion protein involved in the conformational change and theformation of PrPβ aggregates.

Acknowledgments

The authors would like to thank Genome Canada and GenomeBritish Columbia for platform funding for the University ofVictoria-Genome BC Proteomics Centre and for financialsupport. The work on the prion protein was supported by agrant from PrioNet Canada. We would also like to thank DarrylHardie for his assistance with data acquisition on the Orbitrap,and Carol E. Parker for her assistance in editing themanuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jprot.2013.11.020.

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