Identification of Rotenone-Induced Modifications in α-Synuclein Using Affinity Pull-Down and Tandem...

Identification of Rotenone-Induced Modificationsin r-Synuclein Using Affinity Pull-Down andTandem Mass Spectrometry

Hamid Mirzaei,† Jeremy L. Schieler,‡ Jean-Christophe Rochet,*,‡ and Fred Regnier*,†

Department of Chemistry and Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University,West Lafayette, Indiana 47907

Parkinson’s disease is a movement disorder that resultsfrom a loss of dopaminergic neurons in the substantianigra. The disease is characterized by mitochondrialdysfunction, oxidative stress, and the presence of “Lewybody” inclusions enriched with aggregated forms of r-sy-nuclein, a presynaptic protein. Although r-synuclein ismodified at various sites in Lewy bodies, it is unclear howsequence-specific posttranslational modifications modu-late the aggregation of the protein in oxidatively stressedneurons. To begin to address this problem, we developedan affinity pull-down/mass spectrometry method to char-acterize the primary structure of histidine-tagged r-sy-nuclein isolated from catecholaminergic neurons. Usingthis method, we mapped posttranslational modificationsof r-synuclein from untreated neurons and neuronsexposed to rotenone, an inhibitor of mitochondrial com-plex I. Various posttranslational modifications suggestiveof oxidative damage or repair were identified in a regioncomprising a 20-residue stretch in the C-terminal part ofthe protein. The results indicate that r-synuclein is subjectto discrete posttranslational modifications in neurons withimpaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine howspecific modifications of r-synuclein contribute to neu-rologic disorders such as Parkinson’s disease.

Parkinson’s disease (PD) is a neurodegenerative disorder thatresults from a progressive loss of dopaminergic neurons from thesubstantia nigra region of the midbrain.1 The postmortem brainsof PD patients are characterized by indices of oxidative stress2,3

and reduced activity of complex I, an enzyme of the mitochondrialelectron transport chain.3-5 A decrease in complex I activity mayprovoke a “leakage” of electrons from the transport chain, leadingto the accumulation of reactive oxygen species (ROS) that damage

proteins, lipids, and DNA.2,3 The substantia nigra may be particu-larly susceptible to pathogenic mechanisms that upregulate ROSbecause dopaminergic neurons contain relatively high levels ofhydrogen peroxide and hydroxyl radicals (•OH) resulting fromthe metabolism and autooxidation of dopamine.4

A neuropathological hallmark of PD is the presence in somesurviving neurons of cytosolic inclusions, named Lewy bodies,6-8

and dystrophic neuronal processes, termed Lewy neurites.9 Lewybodies and Lewy neurites are both highly enriched with fibrillarforms of R-synuclein,9-11 a 140-amino acid presynaptic protein thatis abundant throughout the brain. The presence of R-synuclein-rich Lewy bodies, Lewy neurites, or both is a characteristic featureof various neurologic disorders in addition to PD, including Lewybody variant of Alzheimer’s disease (LBV) and dementia withLewy bodies (DLB).12 Collectively, diseases involving the aggrega-tion of R-synuclein are referred to as “synucleinopathies”.13

R-Synuclein adopts a “natively unfolded” structure in diluteaqueous solution 14 or an R-helical structure when bound to lipidvesicles.15-19 In contrast, fibrillar R-synuclein has a predominantlyâ-sheet secondary structure.20 The molecular mechanism underly-

* To whom correspondence should be addressed. E-mail: [email protected]; [email protected].

† Department of Chemistry.‡ Department of Medicinal Chemistry and Molecular Pharmacology.

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197.(4) Greenamyre, J. T.; Sherer, T. B.; Betarbet, R.; Panov, A. V. IUBMB Life

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Anal. Chem. 2006, 78, 2422-2431

2422 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006 10.1021/ac051978n CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 02/22/2006

ing the conversion of R-synuclein from natively unfolded orR-helical structures to â-sheet-rich fibrils in Lewy bodies or Lewyneurites is poorly understood.

Data from several studies indicate that aggregated R-synucleinin Lewy bodies or other synucleinopathy inclusions are posttrans-lationally modified at specific residues. Using antibodies that targetnitrated tyrosine residues of R-synuclein, Giasson et al.13 identifiedsites of tyrosine nitration on aggregated R-synuclein from patientswith PD, DLB, LBV, and multiple system atrophy. In addition,Iwatsubo et al.21 showed that R-synuclein is abundantly phospho-rylated at serine 129 in synucleinopathy inclusions. The samegroup also showed that a portion of the phosphorylated, ag-gregated R-synuclein is mono- and diubiquitylated.22 It is notknown whether these posttranslational modifications promoteneurodegeneration or R-synuclein aggregation associated with thesynucleinopathy diseases or whether they are epiphenomena thatplay a minor role in pathogenesis.

Studies with cellular and animal models have begun to shedlight on the role of oxidative stress and R-synuclein aggregationin the pathogenesis of PD. Several groups have shown that rodentsor primates treated with complex I inhibitors such as paraquat,23

MPTP,24-26 or rotenone27 develop a Parkinsonian phenotype,including dopamine neuron death and the presence of cytosolic,R-synuclein-rich inclusions. In addition, Sherer et al.28 reportedthat aggregates enriched with R-synuclein are formed in neuro-blastoma cells chronically treated with low levels of rotenone.Complex I inhibitors may induce neurotoxicity by triggering anincrease in the levels of harmful ROS.27,29,30 Consistent with thisidea, the toxic effects of rotenone in cell culture31 or organotypicslices32 are suppressed by antioxidants. The ROS induced bycomplex I inhibitors are thought to cause posttranslationalmodifications of R-synuclein that promote its conversion to toxicaggregates.13,33-35 However, the nature of these modifications ispoorly understood.

In this study, we describe a novel approach to analyzeposttranslational modifications of R-synuclein in oxidatively stressed

neurons. The method consists of (i) isolating histidine-taggedR-synuclein from stably transfected, catecholaminergic neuronsexposed to rotenone using an affinity pull-down approach and (ii)identifying sequence-specific posttranslational modifications on thehistidine-tagged protein via trypsin digestion coupled with tandemmass spectrometry. Using this method, we have identified adiscrete number of posttranslational modifications in the C-terminal 1/5th of R-synuclein isolated from rotenone-treatedneurons.

EXPERIMENTAL SECTIONMaterials. Dulbecco’s modified Eagle’s medium (DMEM),

horse serum, rotenone, phosphate-buffered saline (PBS), Tris,HEPES, 2-mercaptoethanol, mammalian protease inhibitor cock-tail, NiCl2, imidazole, dithiothreitol (DTT), trypsin, N-R-tosyl-L-lysine chloromethyl ketone (TLCK), myeloperoxidase, and 3-nitro-L-tyrosine were obtained from Sigma Chemical Co. (St. Louis,MO). Urea, NaCl, NaH2PO4, Na2HPO4, CaCl2, NaNO2, and aceticacid were purchased from Mallinckrodt (St. Louis, MO). Thetrichloroacetic acid (TCA) was purchased from VWR International(Chicago, IL). The H2O2 was obtained from Fisher Scientific(Chicago, IL). The acetone was purchased from EMD Biosciences(San Diego, CA). The fetal bovine serum (FBS), penicillin/streptomycin, Lipofectamine 2000, Optimem, and trypsin-EDTAwere obtained from Invitrogen (Carlsbad, CA). Primers for thepolymerase chain reaction (PCR) were purchased from Sigma-Genosys (The Woodlands, TX), and the restriction enzymes NheIand EcoR V were obtained from New England Biolabs (Beverly,MA). ZipTip pipet tips were purchased from Millipore Corp.(Bedford, MA). Tissue culture dishes were purchased from Nunc(Rochester, NY). The PC12 Tet-Off cell line, pTRE2-hyg mam-malian expression vector, doxycycline, and Talon affinity resinwere obtained from BD Biosciences (Mountainview, CA). Hygro-mycin B was purchased from A. G. Scientific (San Diego, CA).

Routine Cell Culture. PC12 Tet-Off cells were cultured in10-cm dishes in PC12 media (DMEM supplemented with 10% (v/v) horse serum, 5% (v/v) FBS, and penicillin/streptomycin). Themedium was changed at 3-day intervals, and the cells werepassaged 1 or 2 times per week.

Preparation of a Mammalian Expression Construct. A 438-bp cDNA encoding R-synuclein with an N-terminal hexahis-tidine tag was amplified by PCR using the following primers:sense primer, 5′-CGAGCTCTCGCTAGCGCCGCCACCATGCAT-CACCATCACCATCACGATGTATTCATGAAAGGACTTTCAAA-GGCC-3′; antisense primer, 5′-CGAGCTCTCGATATCTTAGGCT-TCAGGTTCGTAGTCTTGATACCC-3′. The amplified DNA wassubcloned into the NheI and EcoRV sites of pTRE2-hyg to yieldthe final construct, pTRE2-hyg-6His-RSyn. The sequence of theR-synuclein-encoding insert in this construct was verified usingan Applied Biosystems (ABI) DNA sequencer.

Production of a Stably Transfected PC12 Cell Line. Tet-Off PC12 neurons were plated in the wells of a 24-well plate andgrown to 70% confluency (∼1.4 × 105 cells/well). The mediumwas replaced with fresh medium containing Optimem, and theneurons were transfected with pTRE2-hyg-6His-RSyn using Lipo-

(21) Fujiwara, H.; Hasegawa, M.; Dohmae, N.; Kawashima, A.; Masliah, E.;Goldberg, M. S.; Shen, J.; Takio, K.; Iwatsubo, T. Nat. Cell Biol. 2002, 4,160-164.

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J. H.; Miller, G. W.; Yagi, T.; Matsuno-Yagi, A.; Greenamyre, J. T. J. Neurosci.2003, 23, 10756-10764.

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Analytical Chemistry, Vol. 78, No. 7, April 1, 2006 2423

fectamine 2000 as described by the manufacturer. After 24 h, themedium was replaced with fresh PC12 medium. Then 24 h later,the medium was removed, and the cells were washed with PBS(136 mM NaCl, 0.268 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2-PO4, pH 7.4), trypsinized, and resuspended in fresh PC12 media.The resuspended cells were diluted in 10-cm dishes containingPC12 media supplemented with doxycycline (1 µg/mL) andhygromycin B (300 µg/mL). Single colonies were isolated fromthe 10-cm dishes using the colony-ring method and transferredto the wells of a 12-well plate. The neurons were propagated inthe absence of doxycycline, and the level of R-synuclein in eachclonal cell line was assessed by Western blotting. The stablytransfected cell line with the highest level of R-synuclein (referredto as PC12-C4) was chosen for subsequent analyses.

Purification of Histidine-Tagged r-Synuclein from PC12-C4 Cells. PC12-C4 cells were grown in 245 × 245 mm dishesto 90-95% confluency (∼1.4 × 109 cells). In some experiments,the neurons were cultured in PC12 media during the entire growthperiod, whereas in other cases, the cells were treated with freshmedia supplemented with rotenone (75 nM) for 12 h prior toharvesting. At the end of the cell growth period, the neurons weredislodged from the dishes by a brief treatment with trypsin-EDTAand harvested by centrifugation. The cells were washed with PBSand lysed in a “lysis buffer” (10 mM Tris HCl, pH 7.4, 5 mM2-mercaptoethanol, 300 mM NaCl, 5 mM imidazole, 10 µg/mLmammalian protease inhibitor cocktail) on ice for 15 min. Thecell lysate was boiled for 15 min, and insoluble material wasremoved by centrifugation (20000g, 5 min). The supernatant wasapplied to 200 µL of Talon cobalt affinity resin preequilibrated withlysis buffer. The resin was washed three times with “wash buffer”(lysis buffer minus 2-mercaptoethanol and protease inhibitorcocktail), and protein bound to the resin was eluted with an“elution buffer” (10 mM Tris HCl, pH 7.4, 500 mM imidazole).The eluted protein was dialyzed into 10 mM Tris HCl, pH 8.0,precipitated with TCA (10%, w/v), washed with ice-cold acetone,and dried by rotary evaporation under vacuum prior to analysisby mass spectrometry.

Oxidation of Recombinant r-Synuclein. Histidine-tagged,recombinant R-synuclein was produced in Escherichia coli BL21-(DE3) as described36 and purified using an immobilized metalaffinity column charged with 100 mM NiCl2 (nickel chelatingSuperflow, Pharmacia Biotech). The histidine-tagged protein (2.5mg) was oxidized in a solution of 100 mM Na[Pi], pH 7.4, 200µM H2O2, 100 µM NaNO2, and 2.75 units myeloperoxidase (1 h,25 °C).37 The oxidized R-synuclein was purified using 200 µL ofTalon cobalt affinity resin and prepared for mass spectral analysisas described above.

Proteolysis. To denature and reduce the protein, urea andDTT were added to the proteins to a final concentration of 6 Mand 5 mM, respectively. The samples were incubated for 1 h at60 °C (because R-synuclein does not have any cysteine residues,the alkylation step was omitted). The protein samples were diluted6-fold in a solution of 50 mM HEPES, pH 8.0, 10 mM MgCl2, 10mM CaCl2. Trypsin was added to a final concentration of 2% (w/

v), and the reaction mixture was incubated at 37 °C for at least 8h. Proteolysis was stopped by adding TLCK (trypsin/TLCK ratioof 1:1, w/w).

Reduction of 3-Nitro-L-tyrosine with DTT. 3-Nitro-L-tyrosinewas treated with DTT in trypsin digestion solution (50 mMHEPES, pH 8.0, and 6 M urea) at 60 °C for 1 h. The reactionmixture was cooled to room temperature and diluted 7-fold with50 mM HEPES (pH 8.0). The reaction mixture was then incubatedat 37 °C overnight and analyzed by mass spectrometry.

Mass Spectrometry. R-Synuclein is a small protein with amolecular mass of 14 451 Da. The tryptic digestion map of thepure protein is rather simple, and therefore, additional separationsteps such as reversed-phase chromatography were not requiredto identify peptides spanning almost the entire amino acidsequence. The digested protein samples were desalted usingMillipore ZipTips. Mass spectral analyses were carried out usinga Sciex QSTAR hybrid LC/MS/MS quadrupole TOF massspectrometer. All spectra were obtained in the positive ion mode.Nanospray ionization mode was used to achieve maximumsensitivity. Peptides identified in each sample were sequenced inMS/MS mode. Because different peptides undergo distinctfragmentation patterns due to differences in charge state andsequence, manual MS/MS was considered the best option to havemaximal control over the fragmentation time and collision energy.Most modified peptides exist in very low concentration and canbe easily ignored by an automated method if their intensity doesnot exceed the intensity specified in the method. Manual controlalso allows selective analysis of suspected peptides at very lowconcentrations. This manual approach also enabled us to conservevaluable samples in case of instrument failure or errors in theacquisition method (in contrast to the alternative approach ofunattended automated analysis).

Database Search of MS/MS Data. The sequences of thepeptides were determined by subjecting the MS/MS results to adatabase search using the program MASCOT. The MS/MS datawere searched against the human proteome database. In MAS-COT, the digestion enzyme was set to trypsin with chymotrypticactivity.38 Because lysine and arginine are targets of oxidativemodification that result in tryptic miscleavages, four such mis-cleavages were queried in the database searches. Oxidized (i.e.,carbonylated) derivatives of lysine, arginine, proline, and threoninewere also selected as potential modifications to be queried in thedatabase search, in addition to the peptide modifications listed inTable 3.39 The MASCOT default values were used for all othervariables.

RESULTSAffinity Purification of r-Synuclein. The goal of our study

was to isolate and identify all posttranslationally modified formsof R-synuclein from oxidatively stressed neurons. We usedrotenone-treated PC12 neurons as a cellular model because (i)PC12 cells are catecholaminergic and, therefore, have propertiesof dopaminergic neurons in the substantia nigra and (ii) rotenoneis a complex I inhibitor whose effects mimic mitochondrialdysfunction in PD. To determine the effects of rotenone on the

(36) Rochet, J. C.; Conway, K. A.; Lansbury, P. T., Jr. Biochemistry 2000, 39,10619-10626.

(37) Souza, J. M.; Giasson, B. I.; Chen, Q.; Lee, V. M.; Ischiropoulos, H. J. Biol.Chem. 2000, 275, 18344-18349.

(38) Vajda, T. Ann. Univ. Sci. Budapestinensis de Rolando Eotvos Nominatae, Sect.Chim. 1972, 13, 69-75.

(39) Requena, J. R.; Chao, C.-C.; Levine, R. L.; Stadtman, E. R. Proc. Natl. Acad.Sci. U.S.A. 2001, 98, 69-74.

2424 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

primary structure of R-synuclein in PC12 cells, we first developedan affinity method to isolate posttranslationally modified R-sy-nuclein from a complex mixture of neuronal proteins. We reasonedthat an immunoprecipitation approach was not optimal in this casebecause posttranslational modifications might alter the structureof the protein to an extent that some forms would no longer berecognized by antibodies specific for R-synuclein. Instead, wechose to isolate a histidine-tagged version of R-synuclein from anengineered PC12 cell line using an immobilized metal affinityresin. In pursuing this method, we made the following assump-tions: (i) his6-R-synuclein was biochemically indistinguishablefrom untagged R-synuclein in the PC12 cells and (ii) the his6-tagwas not posttranslationally modified in a way that would precludebinding to the immobilized metal affinity resin. To produce theengineered PC12 cell line (referred to as PC12-C4), Tet-Off PC12cells were stably transfected with a construct encoding R-synucleinwith an N-terminal histidine tag. A lysate from the stablytransfected cells (grown in the absence of doxycycline and in theabsence or presence of rotenone; see below) was boiled toprecipitate unwanted proteins and enrich the sample for R-sy-nuclein, which remains soluble at high temperatures. R-Synucleinwas isolated from the boiled lysate using a cobalt affinity resin.Analysis of the eluted protein by SDS-PAGE with silver stainingindicated that the protein was at least 95-98% pure (data notshown).

Identification of Tryptic Peptides of r-Synuclein fromUntreated Neurons. The first step in our study was to identifyposttranslational modifications on R-synuclein isolated from un-treated PC12-C4 cells. The affinity-purified protein was digestedwith trypsin, and the resulting tryptic peptides were analyzed viatandem mass spectrometry (MS/MS). The MS/MS spectra wereacquired manually rather than with independent data acquisitionmethods to allow more control over the fragmentation of thetryptic peptides. Because the collision energy and collision timecan be adjusted according to the peptide mass and charge to

optimize fragmentation, this manual approach generally resultsin higher scores during database searches. Based on previouslyreported R-synuclein modifications,21,40-42 the following modifica-tions were selected for in the database search: (i) methionineoxidation to methionine sulfoxide or methionine sulfone, (ii)tyrosine modification to nitrotyrosine or aminotyrosine, and (iii)phosphorylation at serine, threonine, or tyrosine.

Next, Mascot was used as the search engine to identify theR-synuclein peptides. In total, 17 peptides accounting for 91% ofthe R-synuclein sequence were identified in the protein samplefrom untreated neurons (Table 1). The only region that was notmapped corresponded to the first 12 residues of R-synuclein,presumably because the peptide from this region contained theengineered histidine tag used for the affinity purification. Onepossible explanation for the absence of this sequence from allmass spectra is a low ionization efficiency. When peptides areexcessively hydrophilic, they cannot escape the droplets in ESI.Increased hydrophilicity of this sequence as a result of themultihistidine tag could result in suppressed ionization.43,44 Fourof the 17 identified peptides appeared as two ions with differentcharges. This redundancy in the data provided additional evidencein support of our assignment of peptide identities. Interestingly,all of the peptides were unmodified except the C-terminal peptidespanning positions 103-140. The modifications identified inR-synuclein from the untreated (control) cells consisted of me-thionine sulfoxide at positions 116 and 127 (Table 1).

A diagram illustrating our analysis of the MS/MS spectrumobtained for the C-terminal peptide with two oxidized methionine

(40) Giasson, B. I.; Lee, V. M. Nat. Neurosci. 2000, 3, 1227-1228.(41) Negro, A.; Brunati, A. M.; Donella-Deana, A.; Massimino, M. L.; Pinna, L.

A. FASEB J. 2002, 16, 210-212.(42) Glaser, C. B.; Yamin, G.; Uversky, V. N.; Fink, A. L. Biochi. Biophys. Acta

2005, 1703, 157-169.(43) Gatlin, C. L.; Turecek, F. J. Mass Spectrom. 2000, 35, 172-177.(44) Flarakos, J.; Xiong, W.; Glick, J.; Vouros, P. Anal. Chem. 2005, 77, 2373-

2380.

Table 1. List of Peptides Identified in r-Synuclein from Untreated (Control) Neurons

no. peptide sequencea location observed mass

1 EGVVAAAEK 13-21 (873.40)1+

(437.20)2+

2 QGVAEAAGK 24-32 (830.40)1+

3 EGVLYVGSK 35-43 (951.60)1+

4 EGVVHGVATVAEK 46-58 (648.40)2+

5 EQVTNVGGAVVTGVTAVAQK 61-80 (964.60)2+

6 E1QVTNVGGAVVTGVTAVAQK 61-80 (650.70)3+

7 TVEGAGSIAAATGF 81-94 (1251.60)1+

8 TVEGAGSIAAATGFVK 81-96 (739.90)2+

9 NEEGAPQEGILEDMPVDPDNEAY 103-125 (1266.60)2+

10 NEEGAPQEGILEDMPVDPD1NEAY 103-125 (1278.10)2+

11 NEEGAPQEGILED1MPVDPD1NEAYEM6PSEEGYQDYEPEA 103-140 (1087.70)4+

12 NEEGAPQEGILEDMPVDPDNEAYEM6PSEEGYQDYEPEA 103-140 (1076.60)4+

(1435.30)3+

13 NEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA 103-140 (1072.70)4+

(1429.70)3+

14 NEEGAPQEGILEDMPVDPDNEAYEM6PSE1EGYQDYEPEA 103-140 (1082.00)4+

15 NEEGAPQEGILEDMPVDPDNEAYE1MPSEEGYQDYEPEA 103-140 (1078.20)4+

(1437.30)3+

16 NEEGAPQEGILEDMPVD1PD1NEAYEM6PSEEGYQDYEPEA 103-140 (1449.70)3+

17 NEEGAPQEGILEDM6PVDPDNEAYE1M6PSEEGYQDYEPEA 103-140 (1447.70)3+

a The modified amino acids are superscripted with a number that indicates the modification type, as follows: D1, sodiated aspartate; E1, sodiatedglutamate; M,6 methionine sulfoxide (see also Table 3). Peptides 7, 9, and 10 are chymotryptic peptides. Their production is due to chymotrypticactivity in the trypsin preparation.38


residues (peptide 17, Table 1) is shown in Figure 1. All of thepeptide fragments identified by Mascot were assigned a labelcorresponding to their m/z value and their ion type (“y” or “b”).The y(13) ion (m/z ) 1513.60 Da) and y(14) ion (m/z ) 1660.63Da) clearly showed the presence of a methionine sulfoxide atposition 127 because the mass difference between these twofragments (147 Da) was equal to the mass of an oxidizedmethionine residue (Figure 1). In contrast, the assignment of amethionine sulfoxide at position 116 was less straightforwardbecause only the y(24)2+ ion (m/z ) 1406.54 Da) was identifiedby Mascot. Mascot assigned 39 of 428 possible fragment ions

using the 92 most intense peaks. However, using less intensepeaks with low signal-to-noise ratios, almost all of the fragmentscould be assigned manually, albeit with a lesser degree ofcertainty. From our manual assignment of peaks, we identifiedthe following ions: b(14), m/z ) 1529.80 Da; b(13), m/z ) 1382.71Da; and y(25)2+, m/z ) 1480.06 Da. The mass difference betweenthe b(14) and b(13) ions and between the y(25)2+ and y(24)2+

ions was 147 Da, indicating that the methionine residue at position116 was oxidized to the sulfoxide. We also reasoned thatmethionine 116 must be oxidized to the sulfoxide because (i) themass of the parent peptide was 32 mass units greater than the

Figure 1. MS/MS analysis of peptide 17 in R-synuclein from untreated (control) neurons. (Top) MS/MS spectrum. All of the y and b ions areassigned in the spectrum. Boxes around the ion labels indicate that the corresponding ions produced a low-intensity signal and were assignedmanually. (Bottom) Diagram showing the y and b ion map superimposed on the peptide sequence, along with calculations of mass differences.Peptide 11 is modified at both of its methionine residues according to the Mascot identification. The oxidized methionine residues are shownwith a star (/) in the sequence.


predicted value and (ii) our data had shown unequivocally that adifference of 16 mass units could be attributed to the oxidation ofmethionine 127. Given that the parent peptide lacked otheroxidizable residues such as histidine or tryptophan, the additionalincrease of 16 mass units must have been due to the oxidation ofmethionine 116 to the sulfoxide.

From all of these data, we concluded that R-synuclein doesnot undergo extensive posttranslational modification in untreatedPC12-C4 cells. The only modifications identified in the protein fromuntreated neurons were two oxidized methionine residues atpositions 116 and 127 in the C-terminal part of the protein.

Identification of Tryptic Peptides of r-Synuclein fromNeurons Treated with Rotenone. The goal of the next set ofexperiments was to determine whether R-synuclein undergoesdifferent posttranslational modifications in oxidatively stressedneurons compared to unstressed cells. Histidine-tagged R-sy-nuclein was affinity purified from PC12-C4 cells treated with themitochondrial complex I inhibitor, rotenone (75 nM, 12 h). Theprotein was digested with trypsin, and the tryptic peptides wereanalyzed by tandem mass spectrometry, as described above. Thetryptic peptides identified in this R-synuclein sample are listed in

Table 2. All of the R-synuclein peptides that were previouslyidentified in the control sample (Table 1) were also found in thesample from the rotenone-treated neurons, with the uniqueexception of the peptide from positions 24-32 in the R-synucleinsequence (24-QGVAEAAGK-32). As in the case of peptides fromthe control sample (Table 1), all of the modifications in the proteinfrom rotenone-treated cells were found in peptides spanningpositions 103-140.

Peptides from the C-terminal region were triply or quadruplycharged and in all but three cases contained at least one sodiatedaspartate or glutamate residue. The peptides were modified invarious combinations, as follows (Table 2): methionine sulfoxide(position 116); phosphotyrosine, nitrotyrosine, or aminotyrosine(position 125); methionine sulfoxide or methionine sulfone (posi-tion 127); nitrotyrosine or aminotyrosine (position 133), andphosphotyrosine or aminotyrosine (position 136). As one example,peptide 11 ((1490.90)3+) in Table 2 contained three differentmodifications: methionine sulfoxide at position 127, nitrotyrosineat position 133, and phosphotyrosine at position 136. Both theMS/MS spectrum and the y and b ion map for this peptide areshown in Figure 2. In this case, Mascot assigned 72 of 428 possible

Table 2. List of Peptides Identified in r-Synuclein from Neurons Treated with 75 nM Rotenone for 12 h

# peptide sequencea location observed mass

1 EGVVAAAEK 13-21 (437.20)2+

2 EGVLYVGSK 35-43 (951.60)1+

3 EGVVHGVATVAEK 46-58 (1295.70)1+

4 EQVTNVGGAVVTGVTAVAQK 61-80 (964.50)2+

5 TVEGAGSIAAATGFVK 81-96 (739.90)2+

6 NEEGAPQEGILEDMPVDPD1NEAYEMPSEEGYQDYEPEA 103-140 (1437.30)3+

7 NEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA 103-140 (1429.70)3+

(1072.40)4+

8 NEEGAPQEGILEDMPVDPDNEAY5EM7PSE1E1GY5QDY5EPEA 103-140 (1470.30)3+

9 NEEGAPQEGILED1M6PVD1PDNEAYEMPSEEGYQDYEPEA 103-140 (1449.60)3+

(1087.50)4+

10 NEEGAPQEGILEDMPVDPDNEAYE1MPSE1EGYQDYEPEA 103-140 (1444.60)3+

11 NEEGAPQEGILEDMPVDPDNEAYEM6PSEE1GY4QD1Y3EPEA 103-140 (1490.90)3+

12 NEEGAPQEGILEDMPVDPD1NE1AYE1MPSEEGYQDYEPEA 103-140 (1451.80)3+

(1088.90)4+

13 NEEGAPQEGILEDM6PVDPDNEAYEMPSEEGYQDYEPEA 103-140 (1435.20)3+

14 NEEGAPQEGILEDMPVDPDNEAYEM6PSEEGYQDYEPEA 103-140 (1076.50)4+

15 NEEGAPQEGILEDMPVDPDNEAY4EMPSEEGYQDYEPEA 103-140 (1444.50)3+(1083.80)4+

16 NEEGAPQEGILEDMPVDPDNE1AYEMPSEEGYQDYEPEA 103-140 (1436.70)3+(1077.90)4+

17 NEEGAPQEGILEDMPVDPDNEAYEM6PSE1EGYQDYEPEA 103-140 (1442.60)3+

18 NEEGAPQEGILED1M6PVD1PD1NE1AY3EM7PSEEGY5QDYEPEA 103-140 (1130.10)4+

19 NEEGAPQEGILEDMPVDPDNEAY5E1MPSEEGYQDYEPEA 103-140 (1081.90)4+

20 NEEGAPQEGILED1MPVDPDNEAY4EMPSEEGY5QDYEPEA 103-140 (1092.90)4+

21 NEEGAPQEGILEDMPVD1PD1NEAY5EM6PSEEGYQDYEPEA 103-140 (1454.90)3+

a The modified amino acids are superscripted with a number that indicates the modification type, as follows: D1, sodiated aspartate; E1, sodiatedglutamate; Y3, phosphotyrosine; Y,4 nitrotyrosine; Y,5 aminotyrosine; M,6 methionine sulfoxide (see also Table 3).

Table 3. List of Amino Acid Modifications Used in Database Searches of the MS/MS Spectra

modifications assigned no.amonoisotopic

mass difference

sodiation of aspartic and glutamic acid 1 21.981 943sodiation of C-terminal amino acid 2 (not found) 21.981 943phosphorylation of tyrosine 3 79.966 331oxidation of tyrosine to nitrotyrosine 4 44.985 078oxidation of tyrosine to aminotyrosine 5 15.010 899oxidation of methionine to methionine sulfoxide 6 15.010 899oxidation of methionine to methionine sulfone 7 31.989 829

a The assigned numbers are used as superscripts to denote modified amino acid residues in Tables 1 and 2.


Figure 2. MS/MS analysis of peptide 11 in R-synuclein from rotenone-treated neurons. (Top) MS/MS spectrum. (Bottom) Diagram showingthe y and b ion map superimposed on the peptide sequence, along with calculations of mass differences. Peptide 11 has three modifications:methionine sulfoxide at position 127, nitrotyrosine at position 133, and nitrotyrosine at position 136. The MS/MS spectrum shows all of thefragments ions needed to calculate the mass of each modified amino acid residue.


fragment ions using the 92 most intense peaks. The assigned yand b ions covered almost the entire sequence including all threemodified amino acids (Figure 2). To confirm the identity of theposttranslational modifications, the mass of the modified aminoacids was calculated from the appropriate y and b ion pairs (Figure2). The mass difference between fragment ions y(14)2+ (m/z )915.28 Da) and y(13)2+ (m/z ) 841.76 Da) matched the mass ofmethionine sulfoxide at position 127. The mass difference betweenfragment ions y*(8) (m/z ) 1144.31 Da) and y(7) (m/z ) 953.29Da) matched the mass of a nitrotyrosine at position 133, and themass difference between fragment ions y0(5) (m/z ) 670.21 Da)and y0(4) (m/z ) 427.18 Da) matched the mass of a phosphoty-rosine at position 136. The tyrosine modifications are significantbecause nitrated tyrosine residues have been identified in fibrillarR-synuclein associated with Lewy bodies,13 whereas evidencesuggests that the phosphorylation of tyrosine 125 inhibits R-sy-nuclein fibrillization.41

A second modified peptide, peptide 8 (1470.30)3+ in Table 2,contained aminotyrosines at positions 125, 133, and 136 and amethionine sulfone at position 127. Both the MS/MS spectrumand the y and b ion map for this peptide are shown in Figure 3.The mass difference between fragment ions b(25)2+ (m/z )1411.08 Da) and b(24)2+ (m/z ) 1329.56 Da) matched the massof a methionine sulfone at position 127. Moreover, the massdifference between fragment ions y(16)2+ (m/z ) 1029.37 Da) andy*(15) (m/z ) 1862.63 Da) matched the mass of an aminotyrosineat position 125, and the mass difference between fragment ionsy(5)2+ (m/z ) 312.14 Da) and y(4) (m/z ) 445.19 Da) matchedthe mass of an aminotyrosine at position 136. We were unable toidentify peptide fragments to obtain direct mass spectral confirma-tion of the presence of aminotyrosine at position 133. However,because the aminotyrosines at positions 125 and 136 wereidentified directly via mass spectral analysis of peptide fragments,the only additional modification that could account for the totalmass of peptide 8 was the presence of an aminotyrosine at position133. The observation that all of the tyrosine residues in this peptidewere converted to aminotyrosine is significant because it suggeststhat nitrated tyrosine residues of R-synuclein may have beenreduced to the corresponding amines in the PC12 cells.

To confirm that aminotyrosines in R-synuclein peptides fromrotenone-treated neurons were not merely artifacts of DTTreduction of nitrotyrosines during tryptic digestion,45 we conductedtwo control experiments. In the first experiment, histidine-taggedrecombinant R-synuclein was treated with myeloperoxidase (knownto modify tyrosine to nitrotyrosine)37 and digested according tothe procedure used for all other samples. We chose to userecombinant R-synuclein from E. coli in this case because ourprevious work indicated that this form of the protein was notmodified at any of its tyrosine residues. Accordingly, it waspossible to nitrate the recombinant R-synuclein on tyrosine withmyeloperoxidase and then test whether the nitrotyrosine wasreduced upon tryptic digestion in the presence of DTT. All of thepeptides derived from the sample were individually subjected toMS/MS analysis, leading to the identification of 22 peptidesencompassing greater than 90% of the R-synuclein sequence.Three oxidative modifications were identified on separate peptides

(methionine sulfoxide and sulfone at position 116; nitrotyrosineat position 125), confirming that the protein was oxidized in thepresence of myeloperoxidase. Importantly, no peptides withaminotyrosine were identified (data not shown). In the secondexperiment, 3-nitro-L-tyrosine was treated with DTT at 60 °C andanalyzed via mass spectrometry. The mass data (not shown)indicated that aminotyrosine was absent from the reaction mixture.The results of both control experiments suggested that aminoty-rosines in the R-synuclein peptides were not generated via DTTreduction, but rather might result from nitrotyrosine reductioninside the cell (see Discussion).

DISCUSSIONA large body of evidence suggests that oxidative stress and

R-synuclein aggregation play an important role in the pathogenesisof PD.1 The postmortem brains of PD patients are characterizedby decreased mitochondrial complex I activity, oxidative damage,and the presence of Lewy bodies enriched with fibrillar R-sy-nuclein.46 Complex I inhibitors elicit increased oxidative stressand R-synuclein aggregation in animal and cell culture models ofPD,27,28 and the aggregation of recombinant R-synuclein is ac-celerated under conditions of oxidative stress.13,33-35 All of theseobservations imply that the conversion of R-synuclein to toxic,aggregated species is accelerated by oxidative damage to theprotein. However, the sequence-specific posttranslational modifica-tions involved in modulating the aggregation of R-synuclein arepoorly defined.

As a first step to understanding the connection betweenR-synuclein oxidation and aggregation, we developed a novelaffinity pull-down/mass spectrometry method to map posttrans-lational modifications of R-synuclein isolated from oxidativelystressed neurons. We chose to use PC12 cells because they arecatecholaminergic, and therefore, they are expected to have highbasal levels of oxidative stress similar to nigral neurons in thebrains of Parkinson’s patients. An important advantage of ourapproach was that the cell lysate was boiled before being appliedto the cobalt affinity resin. In this way, we capitalized on theobservation that R-synuclein is natively unfolded and remainssoluble at high temperatures,14 whereas most other proteinsprecipitate. By combining the protein boiling and affinity pull-downsteps, we were able to isolate histidine-tagged R-synuclein fromthe PC12 cells in a highly purified form. The high purity of theR-synuclein preparations ensured that the mass spectra containedfew contaminant peaks, so that weakly abundant R-synucleinpeptides could be detected and characterized.

The affinity pull-down/mass spectrometry method provided apowerful approach to determine the effects of rotenone treatmenton the primary structure of R-synuclein in PC12 cells. Rotenonewas considered a relevant toxin because it is a complex I inhibitorthat triggers mitochondrial dysfunction and oxidative stress,features characteristic of nigral neurons in the brains of Parkin-son’s patients. Rotenone may play a role in the relatively highincidence of PD in rural areas because of its use in some pesticideformulations.40 In addition, rats treated chronically with low dosesof rotenone develop Parkinsonian symptoms, including motordysfunction, selective dopamine neuron death, and the presenceof Lewy-like inclusions enriched with fibrillar R-synuclein.27

(45) Balabanli, B.; Kamisaki, Y.; Martin, E.; Murad, F. Proc. Natl. Acad. Sci. U.S.A.1999, 96, 13136-13141. (46) Beal, M. F. Ann. N. Y. Acad. Sci. 2003, 991, 120-131.


The only modifications identified in R-synuclein from untreatedPC12 cells were methionine sulfoxides at positions 116 and 127.Peptides with unoxidized methionine residues at these positionswere also identified, suggesting that R-synuclein consists of amixture of modified and unmodified species in untreated neurons.

Although we cannot exclude the possibility of methionine oxida-tion during the protein isolation and tryptic digestion, it is likelythat methionine residues 116 and 127 of R-synuclein were partiallyoxidized in the PC12 cells, given the high levels of oxidative stressin these catecholaminergic neurons. A cycle involving limited

Figure 3. MS/MS analysis of peptide 8 in R-synuclein from rotenone-treated neurons. (Top) MS/MS spectrum. (Bottom) Diagram showing they and b ion map superimposed on the peptide sequence, along with calculations of mass differences. Peptide 8 has four modifications: methioninesulfone at position 127 and aminotyrosine at positions 125, 133, and 136. The mass of each modified amino acid residue was confirmed usingmanually identified fragments.


oxidation of R-synuclein methionine residues followed by enzy-matic repair of the methionine sulfoxides by methionine sulfoxidereductases may have the net effect of preventing a buildup ofROS.42 Therefore, the presence of oxidized methionine residuesin the R-synuclein from untreated cells may reflect an antioxidantdefense mechanism that is active in catecholaminergic neuronseven under basal conditions.42

In the R-synuclein from rotenone-treated neurons, we foundthat methionine was partially oxidized to the sulfoxide at position116 and to the sulfoxide or sulfone at position 127. Our identifica-tion of methionine sulfone in peptides from rotenone-treated butnot untreated cells suggests that R-synuclein undergoes enhancedmethionine oxidation in oxidatively stressed neurons with im-paired complex I activity. We predict that, at high rates ofmethionine oxidation, the hypothetical cellular protective mech-anism outlined above would no longer be effective because theoxidized methionine residues of R-synuclein would not be repairedsufficiently rapidly. Under these conditions, methionine oxidizedforms of R-synuclein would be expected to accumulate. The resultsof test tube studies indicate that methionine oxidized R-synucleinforms aggregates in mixtures with the unmodified protein34 andundergoes fibrillization in the presence of metals.47 Consistent withthese data, increased levels of high molecular weight R-synucleinspecies are observed in rotenone-treated versus untreated PC12-C4 cells (Schieler et al., manuscript in preparation).

In addition to the oxidized methionine residues, we identifiednitrotyrosine, phosphotyrosine, and aminotyrosine in variouscombinations in peptides from the rotenone-treated neurons.Peptides with the corresponding unmodified residues were alsoidentified in the same sample, suggesting that multiple modifiedisoforms of R-synuclein coexist with the native protein in rotenone-treated neurons. Previous studies have shown that (i) tyrosineresidues of R-synuclein are nitrated in Lewy bodies associatedwith PD and other synucleinopathies13 and (ii) recombinantR-synuclein modified by nitration forms soluble oligomers.48,49

Consistent with these reports, our identification of nitrotyrosinein peptides from rotenone-treated PC12 cells suggests that thenitration of tyrosine residues of R-synuclein in neurons withimpaired complex I activity may play a role in pathogenesis. Theidentification of phosphotyrosine and aminotyrosine is remarkablebecause to our knowledge, these modifications have not beenreported in previous studies of R-synuclein in cultured neuronsor brain tissue. The presence of a phosphotyrosine at position136 near a nitrotyrosine at position 133 (peptide 11) is consistent

with previous reports that nitrotyrosine can stimulate nearbytyrosine phosphorylation.50,51 Although a previous report41 indi-cated that tyrosine phosphorylation inhibits R-synuclein fibrilli-zation, this inhibitory effect may lead to the stabilization ofpotentially toxic R-synuclein oligomers in the same way as otherposttranslational modifications.34,35,48,49 We speculate that theaminotyrosines at positions 125, 133, and 136 may have resultedfrom the reduction of nitrotyrosines at these positions.45,52,53 Theresults of our control experiments rule out the possibility that theaminotyrosines were generated via the reduction of nitrotyrosineduring the trypsin digestion. Instead, we propose (as one pos-sibility) that nitrotyrosine may have been reduced enzymaticallyas part of a cellular defense mechanism aimed at eliminatingpotentially toxic, nitrated R-synuclein species. Our observation thattyrosine 136 was only phosphorylated or aminated suggests thatthe enzymatic reduction of a nitrotyrosine at this position mayhave been critical for cell viability.

All of the modifications in R-synuclein from rotenone-treatedneurons occurred at 5 specific residues (M116, Y125, M127, Y133,Y136) spanning a 20-residue region in the C-terminal part of thepeptide. Within this region, tyrosine 125 was phosphorylated,nitrated, or aminated, suggesting that this residue is highlysusceptible to various modifications in oxidatively stressed neu-rons. In addition, tyrosine 133 was nitrated or aminated but neverphosphorylated, indicating that this residue was not targeted bykinases. Evidence from several studies21,22 suggests that serine129, also found in this 20-residue stretch, is phosphorylated infibrillar R-synuclein inclusions associated with Lewy bodies.However, we were not able to identify a peptide with phospho-serine at position 129, perhaps because only part of the R-synucleinfibrillization pathway is reproduced in PC12 cells under theconditions of our study. The fact that all of the modificationsidentified in our study occurred within such a short span in theC-terminal region of the polypeptide chain is remarkable, consid-ering that R-synuclein is natively unfolded.14 Perhaps the N-terminal repeat region of the protein was relatively inaccessibleto posttranslational modifications due to its interaction withmembranes.15-17 Alternatively, oxidative modifications betweenresidues 116 and 136 may have been favored by the binding ofmetals to the abundant acidic residues in this region.35,54 Currentefforts in our laboratory are aimed at assessing how specificmodifications in this 20-residue stretch influence the membranebinding, aggregation, and toxicity of R-synuclein.

In conclusion, we have developed a novel affinity pull-down/mass spectrometry method to identify posttranslational modifica-tions of R-synuclein in oxidatively stressed neurons. This methodis powerful tool to explore how sequence-specific modificationscontribute to the pathogenesis of PD and other synucleinopathies.

ACKNOWLEDGMENTThis work was supported by grants GM59996 and AG13319

from the National Institutes of Health (F.R.) and an award fromthe Showalter Trust (J.-C.R.).

Received for review November 6, 2005. Accepted January30, 2006.

AC051978N

(47) Yamin, G.; Glaser, C. B.; Uversky, V. N.; Fink, A. L. J. Biol. Chem. 2003,278, 27630-27635.

(48) Norris, E. H.; Giasson, B. I.; Ischiropoulos, H.; Lee, V. M. J. Biol. Chem.2003, 278, 27230-27240.

(49) Yamin, G.; Uversky, V. N.; Fink, A. L. FEBS Lett. 2003, 542, 147-152.(50) Brito, C.; Naviliat, M.; Tiscornia, A. C.; Vuillier, F.; Gualco, G.; Dighiero,

G.; Radi, R.; Cayota, A. M. J. Immunol. (Baltimore, Md.: 1950) 1999, 162,3356-3366.

(51) Li, X.; De Sarno, P.; Song, L.; Beckman, J. S.; Jope, R. S. Biochem. J. 1998,331 (Pt 2), 599-606.

(52) Kamisaki, Y.; Wada, K.; Bian, K.; Balabanli, B.; Davis, K.; Martin, E.; Behbod,F.; Lee, Y.-C.; Murad, F. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11584-11589.

(53) Chen, H.-J. C.; Chen, Y.-M.; Chang, C.-M. Chem.-Biol. Interact. 2002, 140,199-213.

(54) Paik, S. R.; Shin, H. J.; Lee, J. H. Arch. Biochem. Biophys. 2000, 378, 269-277.


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