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COMMUNICATIONS

DOI: 10.1002/cbic.200((will be filled in by the editorial staff))

(Autoproteolytic fragments of alpha-synuclein by ion mobility-MS)

Autoproteolytic Fragments are Intermediates in the Oligomerization- Aggregation of Parkinson’s Disease Protein Alpha-Synuclein as Revealed by Ion Mobility Mass Spectrometry

Camelia Vlad,[a] Kathrin Lindner,[a] Christiaan Karreman,[b] Stefan Schildknecht,[b] Marcel Leist,[b] Nick Tomczyk,[c] John Rontree,[c] James Langridge,[c] Karin Danzer,[d] Thomas Ciossek,[d] Alina Petre,[a,e] Michael L. Gross,[e] Bastian Hengerer,[d] and Michael Przybylski[a]*

Several neurodegenerative diseases are characterized by the formation and accumulation of “misfolded” polymeric protein aggregates[1-3]. The formation of neurotoxic oligomers is generally thought to precede aggregation, as shown for ß-amyloid polypeptide (Aβ), Tau- protein and the key protein in Parkinson’s disease (PD), alpha-Synuclein[4-6]. Alpha-Synuclein (αSyn), a 140-aa protein occurring in the presynaptic terminals of neurons, is strongly correlated with PD. αSyn is natively unfolded but adopts an α-helical structure when it binds to lipid vesicles, and forms a β-sheet that facilitates the formation of aggregated morphologies[7,8]. Intracellular accumulation of αSyn aggregates has been recognized at conditions inducing PD[8,9]. Although oligomeric intermediates may represent major neurotoxic species[9], chemical structures of αSyn oligomers and their possible intermediates have not been hitherto identified. The slow rates of formation and low concentrations of aggregating intermediates[9] may be major reasons for the failure of conventional methods of mass spectrometry (MS) to detect and identify oligomers.

MS is now established in biochemistry and structural

biology; for example, electrospray (ESI)-MS[10] has proven highly successful for the structure determination of biopolymers from biological samples[11]. In applications to mixtures, however, low-concentration intermediates and oligomers in αSyn aggregation have not been identified by HPLC-MS, although oligomeric components were observed by gel electrophoresis, atomic force microscopy (AFM), and electron paramagnetic resonance (EPR)[12-14].

Ion mobility mass spectrometry (IMS-MS) is now emerging as a new tool for the analysis of molecular assemblies of proteins[15-18]. The ability of IMS-MS to separate ions as they pass through an electric drift field allows components in protein mixtures to be separated and differentiated according to their size, charge and conformation-dependent topography, as determined by differences in their collisional cross-sections[16-18]. In applications of IMS-MS to oligomerization-aggregation mixtures of αSyn in vitro, we report here the first identification of specific autoproteolytic truncation and degradation products that were previously observed by gel electrophoresis, but not identified. In particular, a highly aggregating fragment was identified by cleavage at the central aggregation domain of αSyn, between residues Val-71 and Thr-72. Aggregation studies of the carboxy-terminal fragment, αSyn(72-140) prepared by both chemical synthesis and recombinant expression, showed a substantially faster fibrillization compared to the intact protein.

The in vitro oligomerization of αSyn was investigated by incubation at 37°C in sodium-phosphate (pH 7.5) for up to 25 days using a previously established procedure[19]. Recombinant αSyn expressed in E. Coli[20] was purified by HPLC to molecular homogeneity, as confirmed by ESI-MS (Figure S1, Supporting Information). The formation of oligomers was monitored by Tris-Tricine PAGE (Figure 1) which revealed bands corresponding to monomeric and oligomer-like αSyn having molecular weights of approximately 17, 35 and 48 kDa. In addition, protein bands with molecular weights lower than full length αSyn were observed with slowly increasing abundances within 2 weeks of incubation, indicating the formation of truncation and/or degradation products. The bands corresponding to αSyn monomer 1a at 17 kDa and dimer 1a’ at 35 kDa were excised from the gel, digested with trypsin and analyzed by HPLC-ESI-MS (Table S2, Supporting Information). Sequencing of the tryptic peptides confirmed that the monomers and dimers of αSyn contain full-length sequences,

[a] Dr. C. Vlad, MSc K. Lindner, Dr. A. Petre, Prof. Dr. M. Przybylski Department of Chemistry

University of Konstanz 78457 Konstanz (Germany) Fax: (+49)7531-88307 E-mail: Michael.przybylski@uni-konstanz.de

[b] Dr. C. Karreman, Dr. S. Schildknecht, Prof. Dr. M. Leist Department of Biology University of Konstanz 78457 Konstanz (Germany)

[c] N. Tomczyk, Dr. J. Rontree, Dr. J. Langridge Waters Ltd., Micromass Atlas Park Manchester, M225UP (UK)

[d] Dr. K. Danzer, Dr. T. Ciossek, Prof. Dr. B. Hengerer ZNS research Böhringer Ingelheim Pharma GmBH & Co KG 88397 Biberach/Riß (Germany)

[e] Dr. A. Petre, Prof. Dr. M. Gross Department of Chemistry Washington University St. Louis St. Louis, Mo 63130 (USA)

Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

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respectively, since all tryptic peptides and no truncated sequences were found. Further structural characterization was obtained by N-terminal Edman sequencing after transfer of the proteins onto a PVDF membrane, which yielded the first 20 amino acids of intact αSyn. Direct ESI-MS analysis of the αSyn incubation mixture after a short time period (3 hrs), however, revealed a small amount of N-terminally truncated αSyn lacking the first 6 amino acids (αSyn(7-140); spot 1b in Figure 1; Table S1 and Figure S2, Supporting Information). In contrast to these results, attempts to identify the degradation products (bands 1c, 1d, 1e in Figure 1) by direct ESI-MS and HPLC-MS were unsuccessful, presumably owing to their low concentrations.

Figure 1. Time-dependent autoproteolytic degradation of αSyn and modified αSyn polypeptides visualized by Coomassie (left) and silver staining (right), at begin of incubation (A), 4 days (B) and 13 days (C). 1, wt-αSyn; 2, recombinant αSyn(Ala6(70-75)); 3, recombinant αSyn(Gly6(70-75)); 4, αSyn(72-140) synthesized by SPPS; 5, recombinant αSyn(72-140); 6, recombinant βSyn. Full length αSyn monomer and dimer are denoted 1a and 1a’, respectively; N-terminally truncated αSyn(7-140) is denoted 1b. αSyn fragments are denoted 1c, 1d, 1e; 1c’ is the dimer of fragment 1c. β-Synuclein 6 lacks the (72-83) sequence. 1 and 1+ denote incubation of wt-αSyn without and with addition of protease inhibitors (serine-, cysteine- proteases; metalloproteases).

The reaction mixtures upon incubation of αSyn for 4 up to 21 days were subjected to ion mobility-MS analysis, which provides inherently three-dimensional data consisting of ion mobility-dependent drift time and separation according to differences in protein collisional cross sections[15,16], mass/charge, and relative abundances. Broad-band admittance to the drift region afforded an ion-mobility plot of the incubation mixture after

7 days and showed separation into two peaks corresponding to multiply charged ion series with different drift times (Figure 2A; Figure S3, Supporting Information). These proteins were identified by molecular mass determinations and ESI-tandem-MS sequencing (Figure 2B-D). Deconvolution of the multiply charged ion series to singly charged ions provided identification of full length αSyn monomer (14459.4 Da) and dimer (28919.6 Da) in peak 2, in agreement with the mass spectrometric identification of the gel electrophoretic bands 1a and 1a’ in Figure 1 (Figure 2C and Table S2, Supporting Information). In contrast to the intact αSyn proteins, the IMS-MS analysis of peak 1 at a decreased drift time revealed proteolytic products by N- and C-terminal truncation, αSyn(14-133) (12162.5 Da) and αSyn(40-140) (10436.4 Da), corresponding to the proteolytic gel bands 1c, 1e; Figure 1 and Table S1, Supporting Information). A most remarkable fragment was identified in the ion series within peak 1 by cleavage at residues Val-71 and Thr-72 in the central amyloidogenic domain αSyn(61-93), corresponding to the gel electrophoretic band 1d (Figure 1). This carboxy-terminal fragment, αSyn(72-140) was identified by (i), accurate mass determination (7274.4 Da) and tandem-MS sequencing in the IMS-MS experiment (Figure 2C, 2D); (ii), moreover, incubation of αSyn for extended time periods (>14 days) afforded formation of this fragment in amounts sufficient for direct elution of the electrophoretic band and identification by MALDI-MS (Table S1, Supporting Information). Additional characterization of the C-terminal fragments αSyn(40-140) and αSyn(72-140) was obtained by affinity-mass spectrometry[21] and Western-Blot analysis with a monoclonal αSyn antibody recognizing a C-terminal αSyn epitope, as identified by proteolytic-excision mass spectrometry[22] (data not shown).

The proteolytic fragment αSyn(72-140) was synthesized by (i), solid-phase peptide synthesis on a semi-automated peptide synthesizer using the Fmoc-strategy with double coupling and capping in the final 30 cycles (band 4), and (ii), recombinant expression in E. Coli[20] (band 5; s. Figure 1). Polypeptides prepared by both methods were purified to homogeneity by semi-preparative HPLC; their molecular masses and sequences agreed with expected values as determined by ESI-MS and tandem-MS sequencing (Figure S4, Supporting Information). Tris-Tricine PAGE of 4 and 5 showed identical major bands at approximately 12 kDa in agreement with the band of the autoproteolytic fragment 1d of intact αSyn; most remarkably, in contrast to intact αSyn PAGE the fragment 5 revealed the rapid formation of oligomers (s. Figure 3A). The in vitro aggregation of αSyn(72-140) 5 was analyzed in comparison with full length αSyn 1 for up to 5 days at 7 - 30 µM concentrations using Thioflavin T (ThT) fluorescence[23] as an established fibrillization assay, and the results compared with the oligomerization analysis using Tris-Tricine PAGE (Figure 3). Although exact monitoring of fibrillization kinetics was hampered by increasing insolubility of 5 at longer aggregation times, the results showed a substantially faster aggregation of αSyn(72-140). In contrast to full-length αSyn, the fragment 5 showed a typical sigmoidal curve characteristic of reactive aggregating polypeptides.

The autoproteolytic degradation of αSyn and significance of the fragment αSyn(72-140) were ascertained by a number of additional mass spectrometric and gel electrophoretic data, and by control experiments with several synuclein polypeptides. Comparative in vitro studies of the non-aggregating brain protein, β-synuclein (βSyn) which lacks the central amyloidogenic domain (72-83), showed neither oligomerization-aggregation, nor any autoproteolytic cleavage within 21 days of incubation (Figure 1,

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6); identical expression systems and HPLC purification procedures were employed for ßSyn and αSyn, thus excluding αSyn degradation by an external protease. Further results providing evidence to exclude a proteolytic cleavage of αSyn by contaminating proteases were: (i), incubation of αSyn with and without addition of a broad spectrum of protease inhibitors yielded identical autoproteolytic degradation (Figure 1; 1+ and 1, right lane); (ii), boiling of samples prior to incubation and use of sterile buffers showed no change of αSyn autoproteolysis; (iii), autoproteolytic degradation was still observed for αSyn mutants 2 and 3 in which the six residues (70-75) were exchanged against Ala and Gly residues (s. Figure 1). Moreover, a recent study on the interaction of αSyn with ß-glucocerebrosidase (GCase), the target enzyme of Gaucher’s disease, in cortical neurons lends additional support to the significance of our results, showing that the neurotoxicity-enhancing effect of αSyn upon depletion of

GCase is abolished in a mutant αSyn lacking the sequence (71-82)[24].

Further support for the importance of the central domain for the autoproteolysis of αSyn was obtained from initial hydrogen-deuterium exchange mass spectrometry (HDX-MS)[25] studies using high resolution ESI-MS. HDX-MS of full length αSyn 1 showed rapid exchange (<3 hrs) for 115 of 134 backbone hydrogens while 19 aa remained resistant to exchange for >14 days (data not shown). Remarkably, an identical number of 19 aa in the αSyn(72-140) fragment 5 was found resistant to HDX (exact determination of the shielded domain is currently performed). These results suggest shielding of a C-terminal part of the hydrophobic amyloidogenic domain within residues 73/74 and 93, and are consistent with autoproteolytic accessibility at residues 71/72.

Figure 2. ESI-IMS- MS identification of full length αSyn and fragment αSyn(72-140) in the incubation mixture of wt- αSyn after 7 days. (A), IMS-MS Drift time vs. m/z of αSyn incubation mixture in PBS buffer (pH 7.5) after seven days at 37 oC. (B), Deconvoluted mass spectrum of ions corresponding to peak 1, proteolytic fragment αSyn(72-140); (C), Deconvoluted mass spectrum of ions corresponding to peak 2 showing full length αSyn monomer and dimer; (D), Tandem-MS sequence determination of αSyn(72-140) , peak 1, showing the partial sequence (101-119).

In conclusion, ion-mobility MS owing to its capability to separate αSyn and its autoproteolytic products in a concentration- independent manner, enabled the characterization of intermediate fragments in the in vitro oligomerization-aggregation; particularly, IMS-MS revealed a possible key fragment, αSyn(72-

140). The yet unknown mechanism of autoproteolysis, possibly proceeding via initial N-terminal truncation products observed, may be amenable by examination of specific functional αSyn residues. Thus, modification of full length αSyn 1 by amino-succinylation[26] provided a stable derivative succinylated at all

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Lys residues and at His50 as confirmed by ESI-MS; this succinylated αSyn did not show any aggregation or fragmentation, and only formation of a dimer (Figure S5, Supporting Information). The detailed biochemical evaluation of autoproteolytic products, presently carried out in our laboratories, may be a key to the elucidation of the oligomerization-aggregation mechanism of αSyn underlying its neurotoxicity. Moreover, the application of IMS-MS is expected to be highly valuable for identifying proteolytic products in the in vivo aggregation of αSyn.

Experimental Section

Gel electrophoresis of αSyn oligomerization in vitro: αSyn oligomers were prepared by incubation of several batches in triplicate using 5 - 30 µM solutions for up to 25 days in sodium phosphate buffer (pH 7.5) as previously described[19]. Proteins were solubilized and denatured using stock solutions of sample buffer containing 4 % SDS, 25 % glycerol, 50 mM Tris-buffer and 6 M urea, pH 6.8; gels were run at 100 V until the tracking dye reached the bottom. Separations were performed by Tris-Tricine PAGE on a Mini-Protean-3 cell (BioRad, München, Germany) using 12-15 % PAGE, and protein bands visualized by Coomassie Blue and Silver staining (90 x 60 x 1 mm gels).

Figure 3. Thioflavin T fibrillization of the αSyn(72-140) fragment 5 (A) compared to intact wt- αSyn 1 (B). Proteins were incubated at 30 µM concentrations for 5 days in 20 mM Na2HPO4 pH 7.5 at 37 oC, and ThT fluorescence determined 450 nm excitation and 486 nm emission (F450/486). The final assay volume (100 µL) contained 25 µM ThT and 7 µM αSyn sample. The inserts show Tris-Tricine-PAGE at the begin of incubation (left) and after 4 days (right) using silver nitrate staining.

For the protease inhibitor mixture (Complete-Mini; Roche Applied Science, Mannheim, Germany) ca. 50 µM was dissolved in 1.5 ml 20 mM phosphate buffer pH 7.5.

Ion mobility mass spectrometry: Ion mobility mass spectrometry was performed on a Synapt-G1 QTOF-mass spectrometer (Waters, Manchester, UK) equipped with an electrospray ionization source. Ions passed through a quadrupole were either set to transmit a substantial mass range or to select a particular m/z before entering the Triwave ion mobility unit comprising a T-Wave trap unit for ion accumulation; stored ions were gated (500 µsec) into the IMS- T-Wave unit for separation according to ion mobilities and passed to the T-Wave unit for transfer into the orthogonal-TOF analyzer[15a] (Figure S3; Supporting Information). Pressures in the T-Wave trap and transfer regions were 7e-2 mbar (Ar) and 0.5 mbar (nitrogen), respectively; sample injection volume was 5 µL. IMS acquisition was performed at 350 - 4000 m/z range, 25 V cone voltage, 0.45 bar IMS pressure, and 5 - 15 V wave height. Sample injection was performed using an Advion Triversa Nanomate as previously described[18a,b].

Synthesis of αSyn polypeptides: Solid phase synthesis of αSyn(72-140) 4 was performed on a semi-automated peptide synthesizer EPS-221 (Intavis, Köln, Germany) using a NovaSyn TGR resin; Fmoc protection was employed with double coupling and capping within residues (73-98), and mass spectrometric monitoring of intermediate crude products[21a, 27]. Fmoc amino acids, NovaSyn TGR resin, and other reagents for activation and protection were obtained from Novabiochem (Laufelfingen, Switzerland). The general protocol applied was: DMF washing followed by deprotection with 20 % piperidine in DMF; 8 min deprotection, 30 min coupling. Coupling was carried out in a mixture solution containing PyBOP and N-methylmorpholine (NMM) in DMF; the resin was then washed with DMF, ethanol and dried under vacuum[27]. Final deprotection of side chains and cleavage from the resin was performed with trifluoroacetic acid / triethylsilane / water (95: 2.5: 2.5, v/v/v) for 2 hrs. Resin and crude peptide were separated by filtration, and the crude peptide subjected to HPLC purification.

Recombinant expression of full length αSyn 1 and αSyn(72-140) 4 was performed using the E. Coli expression system BL 21 (DE3) [pLys] strain and the T7 RNA polymerase system. Harvested cells were centrifuged, resuspended in PBS and heated for 2 min to 100 °C; the cell suspension was centrifuged for 15 min at 4300 rcf and the protein redissolved in PBS buffer. HPLC purification of synthetic and recombinant αSyn polypeptides was performed with a BioRad-3000 semipreparative system (BioRad, Richmond, CA) on a Vydac-C4 column (250 x 4.6 mm) with a linear gradient (0.1 % TFA / 0.1 % TFA / 80 % acetonitrile) at a flow rate of 1 mL/min (Figure S4; Supporting Information).

Thioflavin-T (ThT) aggregation: Purified αSyn 1 and αSyn(72-140) 5 were dissolved in 20 mM Na2HPO4 containing 0.03 % NaN3 (pH 7.5) at 7 up to 30 µM in microcentrifuge tubes, and triplicates incubated at 37 °C with agitation. For ThT assay a 23 µL sample was withdrawn at regular time intervals. The final assay volume (100 µL) contained 25 µM ThT/7 µM protein; a blank of 25 µM ThT was used as a control. Determinations were performed in 96-well MTP with a Victor-2 fluorescence plate reader (Perkin-Elmer, Überlingen, Germany) at 450 nm excitation and 486 nm emission. Data were analyzed with Graph

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Pad Prism wherein the sigmoidal increase of the ThT fluorescence was analyzed.

Acknowledgements

This work was supported by the Konstanz Research Graduate School Chemical Biology, and the Preoteostasis Research Center, University of Konstanz; and The Landesstiftung für Wissenschaft und Forschung Baden-Württemberg.

Keywords: Alpha-synuclein · oligomerization-aggregation · ion mobility mass spectrometry · autoproteolytic fragmentation · in vitro aggregation

[1] (a) M. S. Forman, V. M. Lee, J. Q. Trojanowski, Neuron 2005, 47,

479. (b), M. Bucciantini, E. Giannoni, F. Chiti, F. Baroni, L. Formigli, J. Zurdo, N.

Taddei, G. Ramponi, C.M. Dobson, M. Stefani, Nature 2002, 416, 507. (c), H.

Olzscha, S.M. Schermann, A.C. Woerner, S. Pinkert, M.H. Hecht, G.G.

Tartaglia, M. Vendruscolo, M. Hayer-Hartl, F.U. Hartl, R.M. Vabulas, Cell 2011

144, 67.

[2] (a) H. Mirzaei, J. L. Schieler, J. C. Rochet, F. Regnier, Anal Chem

2006, 78, 2422. (b) A. Salminen, J. Ojala, A. Kauppinen, K. Kaarniranta, T.

Suuronen, T. Prog. Neurobiol. 2009, 87, 181. (c), A.K. Dunker, I. Silman, V.N.

Uversky, J.L. Sussman, Curr. Opin. Struct. Biol. 2008, 18, 756.

[3] (a) T. M. Dawson, V. L. Dawson, Science 2003, 302, 819. (b) B.

Tolnay, T. Probst Neuropathology and Applied Neurobiology 1999, 25, 171.

[4] (a) M. H. Polymeropoulos, C. Lavedan, E. Leroy, S. E. Ide, A.

Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E. S. Stenroos, S.

Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W. G. Johnson, A.

M. Lazzarini, R. C. Duvoisin, G. Di Iorio, L. I. Golbe, R. L. Nussbaum, Science

1997, 276, 2045. (b) M. Goedert, Nat. Rev. Neuroscience 2001, 2, 492. (c) C.W.

Shults C.W., Proc. Natl. Acad. Sci. USA 2006, 103, 1661.

[5] (a) R. Kruger, W. Kuhn, T. Muller, D. Woitalla, M. Graeber, S.

Kosel, H. Przuntek, J. T. Epplen, L. Schols, O. Riess, Nat Genet 1998, 18, 106.

(b), V.N. Uversky, J. Neurochem. 2007, 103, 17.

[6] J. J. Zarranz, J. Alegre, J. C. Gomez-Esteban, E. Lezcano, R. Ros,

I. Ampuero, L. Vidal, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, E.

Gomez Tortosa, T. del Ser, D. G. Munoz, J. G. de Yebenes, Ann Neurol 2004,

55, 164.

[7] A. R. Saha, J. Hill, M. A. Utton, A. A. Asuni, S. Ackerley, A. J.

Grierson, C. C. Miller, A. M. Davies, V. L. Buchman, B. H. Anderton, D. P.

Hanger, J Cell Sci 2004, 117, 1017.

[8] R. Kayed, Y. Sokolov, B. Edmonds, T. M. McIntire, S. C. Milton, J.

E. Hall, C. G. Glabe, J Biol Chem 2004, 279, 46363.

[9] (a) J. Li, V.N. Uversky, A.L. Fink Neurotoxicology 2002, 23, 553.

(b) L. Crews, I. Tsigelny, M. Hashimoto, E. Masliah, E. Neurotox. Res. 2009, 16,

306.

[10] J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse,

Science 1989, 246, 64.

[11] (a) A. J. Heck, R. H. Van Den Heuvel, Mass Spectrom Rev 2004,

23, 368. (b) M. Przybylski, M.O. Glocker, Angew. Chem. Int. Ed. Engl. 1996, 35,

806. (c) P. Kebarle, U.H. Verkerk, Mass Spectrom. Rev. 2009, 28, 898. (d), P.

Nemes, S. Goyal, A. Vertes, Anal. Chem. 2007, 80, 387.

[12] (a), W. Kim, Y. Kim, J. Min, D.J. Kim, Y.T. Chang, M.H. Hecht,

ACS Chem. Biol. 2006, 1, 461. (b) A. Görg, G. Boguth, C. Obermaier, A. Posch,

W. Weiss, Electrophoresis 1995, 16, 1079. (c) H. Schagger, Nat. Protoc. 2006,

1, 16.

[13] (a), W.S. Gosal, S.L. Myers, S.E. Radford, N.H. Thomson, Protein

Pept Lett, 2006, 13, 261-270. (b), Z. Wang, C. Zhou, C. Wang, L. Wan, X. Fang,

C. Bai, Ultramicroscopy 2003, 97, 73-79. (c), M. Drescher, F. Godschalk, G.

Veldhuis, B.D. van Rooijen, V. Subramaniam, M. Huber, Chembiochem. 2008,

9, 2411.

[14] (a) I. Bharathi, K.S. Rao, Neurosci. Lett. 2007, 424, 78. (b), H.A.

Lashuel, J.S. Wall, Methods Mol. Biol. 2005, 299, 81. (c), A.C. Ferreon, Y.

Gambin, E.A. Lemke, A.A. Deniz, Proc. Natl. Acad. Sci. USA 2009, 106, 5645.

[15] (a) S.D. Pringle, K. Giles, J.L. Wildgoose, J.P. Williams, S.E.

Slade, K. Thalassinos, R.H. Bateman, M.T. Bowers, J.H. Scrivens, Int. J.Mass

Spectrom. 2007, 261, 1. (b) A.B. Kanu, P. Dwivedi, M. Tam, L. Matz, H.H. Hill, J.

Mass Spectrom. 2008, 43, 1. (c) C. Bleiholder, N.F. Dupuis, T. Wyttenbach, M.T.

Bowers, Nat. Chem. 2011, 3, 172. (d) B.T. Ruotolo, J. L. Benesch, A. M.

Sandercock, S. J. Hyung, C. V. Robinson, Nat Protoc. 2008, 3, 1139. (e) C.

Uetrecht, R.J. Rose, E. van Duijn, K. Lorenzen, A.J. Heck, Chem. Soc. Rev.

2010, 39, 1633.

[16] (a) C. A. Srebalus, J. Li, W. S. Marshall, D. E. Clemmer, Anal

Chem 1999, 71, 3918. (b) S. Trimpin, D. E. Clemmer, Anal Chem 2008, 80,

9073. B. T. Ruotolo, S. J. Hyung, P. M. Robinson, K. Giles, R. H. Bateman, C. V.

Robinson, Angew Chem Int Ed Engl 2007, 46, 8001.

[17] (a) B.T. Ruotolo, S.J. Hyung, P.M. Robinson, K. Giles, R.H.

Bateman, C.V. Robinson, Angew Chem Int Ed Engl 2007, 46, 8001. (b) M. Zhou,

A.M. Sandercock, C.S. Fraser, G. Ridlova, E. Stephens, M.R. Schenauer, T.

Yokoi-Fong, D. Barsky, J.A. Leary, J.W. Hershey, Proc. Natl. Acad. Sci. USA

2008, 105, 18139.

[18] (a) M. Kloniecki, A. Jablonowska, J. Poznanski, J. Langridge, C.

Hughes, I. Campuzano, K. Giles, M. Dadlez, J. Mol. Biol. 2011, 407, 110. (b) M.

Iurascu, C. Cozma, N. Tomczyk, J. Rontree, M. Desor, M. Drescher, M.

Przybylski Anal. Bioanal. Chem. 2009, 395, 2509. (d) M. Iurascu, C. Cozma, J.

Langridge, N. Tomczyk, M. Desor, M. Leist, M. Przybylski, In: C.I. Wilkins and S.

Trimpin (Eds.) Ion Mobility Spectrometry - Mass Spectrometry: Theory and

Applications. 2011, in press. (e) Y. Liu, L.H. Ho, J.A. Carver, T.L. Pukala, T.L.,

Austral. J. Chem.2011, 64, 36.

[19] K.M. Danzer, D. Haasen, A.R. Karow, S. Moussaud, M. Habeck,

A. Giese, H. Kretzschmar, B. Hengerer, M. Kostka, J Neurosci 2007, 27, 9220.

[20] K. Ueda, H. Fukushima, E. Masliah, Y. Xia, A. Iwai, M. Yoshimoto,

D.A. Otero, J. Kondo, Y. Ihara, T. Saitoh Proc. Natl. Acad. Sci. USA 1993, 90,

11282.

[21] (a) A. Marquardt, S. Muyldermans, M. Przybylski Chemistry 2006,

12, 1915. (b) M. Macht, A. Marquardt, S.O. Deininger, E. Damoc, M. Kohlmann,

M. Przybylski, Anal. Bioanal. Chem. 2004, 378, 1102. (c) M. Dragusanu, B. A.

Petre, S. Slamnoiu, C. Vlad, T. Tu, M. Przybylski, J. Am. Soc. Mass Spectrom.

2010, 21, 1643.

[22] (a) D. Suckau, J. Kohl, G. Karwath, K. Schneider, M. Casaretto, D.

Bitter-Suermann, M. Przybylski, Proc. Natl. Acad. Sci. USA 1990, 87, 9848. (b)

P. Juszczyk, G. Paraschiv, A. Szymanska, A.S. Kolodziejczyk, S. Rodziewicz-

Motowidlo, Z. Grzonka, M. Przybylski J. Med. Chem. 2009, 52, 2420. (c) J.

McLaurin, R.E. Cecal, M. Kierstead, X. Tian, A. Phinney, M. Manea, M. French,

M. Lambermon, A. Darabie, M. Brown, C. Janus, C. Chishti, P. Horne,

D.Westaway, P. Fraser, H. Mount, M. Przybylski, P. St George-Hyslop Nature

Med. 2002, 8, 1263-1269. (d) R. Stefanescu, R.E. Iacob, E. Damoc, A.

Marquardt, E. Amstalden, M. Manea, I. Perdivara, M. Maftei, G. Paraschiv, M.

Przybylski Eur. J. Mass Spectrom. 2007, 13, 69.

[23] R. Khurana, C. Coleman, C. Ionescu-Zanetti, S.A. Carter, V.

Krishna, R.K. Grover, R. Roy, S. Singh, J. Struct. Biol. 2005, 151, 229.

[24] J.R. Mazzulli, Y.H. Xu, Y. Sun, A.L. Knight, P.J. McLean, G.A.

Caldwell, E. Sidransky, G.A. Grabowski, D. Krainc, Cell 2011, 146, 37.

[25] (a), I. Kheterpal, M. Chen, K.D. Cook, R. Wetzel, J. Mol. Biol. 2006,

361, 785. (b), M.M.Zhu, D. Rempel, Z. Du, M.L. Gross, J. Am. Chem. Soc. 2003,

125, 5252.

[26] M. Przybylski, M. Glocker, U. Nestel, V. Schnaible, M. Blüggel, K.

Diederichs, J. Weckesser, M. Schad, A. Schmid, W. Welte, R. Benz Protein Sci.

1996, 5, 1477.

[27] J.E. Jung, H.P. Wollscheid, A. Marquardt, M. Manea, M. Scheffner,

M. Przybylski Bioconjug. Chem. 2009, 20, 1152-1162.

Received: ((will be filled in by the editorial staff))

Published online: ((will be filled in by the editorial staff))

6

COMMUNICATIONS

Ion mobility- mass spectrometry of the in vitro

oligomerization-aggre-gation of Parkinson’s disease protein, alpha-synuclein, revealed auto-proteolytic products - particularly, a highly aggregating C-terminal fragment, αSyn(72-140).

Camelia Vlad, Kathrin Lindner, Christiaan Karremann, Stefan Schildknecht, Marcel Leist, Nick Tomczyk, John Rontree, James Langridge, Karin Danzer, Thomas Ciossek, Alina Petre, Michael Gross, Bastian Hengerer and Michael Przybylski

Page No. – Page No.

Autoproteolytic Fragments are Intermediates in the Oligomerization-Aggregation of Parkinson’s Disease Protein Alpha-Synuclein as Revealed by Ion Mobility Mass Spectrometry

7

Supporting Information

Autoproteolytic Fragments are Intermediates in the Oligomerization-Aggregation of Parkinson’s

Disease Protein Alpha-Synuclein as Revealed by Ion Mobility Mass Spectrometry

Camelia Vlad, Kathrin Lindner, Christiaan Karreman, Stefan Schildknecht, Marcel Leist, Nick Tomczyk, John Rontree, James Langridge, Karin Danzer, Thomas Ciossek, Bastian Hengerer and Michael Przybylski

Materials and Methods

HPLC-MS analysis of αSyn oligomerization products

ESI-ion trap mass spectra were recorded on a Bruker Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen,

Germany) equipped with a direct infusion and ESI-LC-MS system. LC-MS experiments were carried out using an Agilent

Technologies (Waldbronn, Germany) HP1100 liquid chromatograph for binary gradient elution (pump model G1312A), including an

autosampler (G1313A) and a DAD (G1315 B) coupled to the ion trap mass spectrometer. A 150 mm x 4.6 mm x 3 μm Discovery

RP-18 column was used for the separation of peptides. Mass spectra were recorded in the full scan mode, scanning from m/z 200 to

1500. Ion source parameters were 20 psi nebulizer gas and 9 L/min of drying gas, with a temperature of 300 °C. Tandem-MS

experiments were carried out in the autofragmentation mode. Sequences of the extracted peptides were determined by subjecting the

tandem- MS results to the NCBInr database search using the MASCOT programme.

Amino-Succinylation of alpha-synuclein

Chemical modification of proteins by amino-succinylation with succinic anhydride has been successfully used in the structure-

function studies of several proteins, by reaction of succinic anhydride with lysine-ε-amino groups and the N-terminal α-amino group,

converting them from basic to acidic protein derivatives[26]. Succinylation of αSyn was performed in 0.3 M sodium phosphate buffer,

pH 7.5, by addition of increasing amounts (1 mg/mL) of a succinic anhydride solution in DMSO (0.5, 1, 2, 10, 50, 100-fold molar

excess to protein) to the continuously stirred protein solution (1 mg/mL) over a period of 90 min, and the pH of the reaction mixture

maintained by addition of 0.2 M NaOH. The reaction was allowed to proceed for 30 min at 25 °C and the protein solution then

thoroughly dialyzed against water, followed by gel electrophoresis and mass spectrometric analysis. As shown in Figure S5A, B, the

succinylation provided a protein derivative with a mass increase of 1700 Da corresponding to modification of all lysine residues (15)

and the N-terminus, and the His50 residue; the fully succinylated αSyn does not show any oligomerization- aggregation, and only a

dimer (Figure S5).

8

Table S1: αSyn Autoproteolytic truncation and degradation products observed by gel electrophoresis and identified by IMS-MS

and MALDI-MS.

No. a Spot No. a m.w. gel [kDa] b

m.w. MS c

[Da] Sample description Sequence g

1 a 16.7 14460.0 d wt-αSyn 1-140

a’ 35.1 28919.6 d wt-αSyn / dimer 1-140

b 16.7 13706.6 e wt-αSyn / N-terminal truncation 7-140

c 14.5 12163.0 d wt-αSyn / N-terminal truncation 14-133

c’ 25.9 25081.5 f wt-αSyn / dimer of N-terminal truncation

d 12.3 7274.5 d,e,f wt-αSyn / autoproteolytic fragment 72-140

e 10.9 10436.0 d wt-αSyn / N-terminal truncation 40-140

2 16.7 14329.4 e αSyn V70A V71A T72A G73A V74A T75A 1-140

3 16.7 14245.4 e αSyn V70G V71G T72G G73G V74G T75G 1-140

4 12.3 7365.3 e αSyn(71-140) / chemical synthesis 71-140

5 12.3 7274.4 e αSyn(72-140) / recombinant 72-140

6 17.4 14288.7 e βsyn 1-140

aSyn sample/spot number corresponding to Figure 1. bMol. weight of bands observed by gel electrophoresis. cMolecular mass of truncation/degradation products determined by mass spectrometry (MS)

dMolecular mass of truncation/degradation products determined by IMS-MS. eMolecular mass of truncation/degradation products determined ESI-MS. fMolecular mass of truncation/degradation products determined MALDI-TOF-MS. gN-Terminal sequence determined by Edman sequencing (Applied Biosystems Procise-470 sequencer).

Table S2: Mass spectrometric characterization of electrophoretic bands: Tryptic peptides of monomer and dimer αSyn 1a and

1a’ identified by a) LC/ESI-MS and b) MALDI-TOF-MS.

No. Peptide sequence Molecular ion observed

Mexp Mcalc

1 AKEGVVAAAEK 11 - 21 a) 536.7)2+ 1071.4 1071.6

2 EGVVAAAEK 13 - 21 a) (437.2)2+ 872.7 872.4

3 EGVVHGVATVAEKTK 46 - 60 a) (509.1)3+ 1523.4 1523.8

4 EGVVHGVATVAEK 46 - 58 a) (432.6)3+ 1295.1 1295.6

5 EGVLYVGSKTK 35 - 45 a) (950.8)2+ 1179.5 1179.6

6 EGVLYVGSK 35 - 43 a) (476.2)2+ 950.5 950.5

7 MDVFMK 1 - 6 a) (385.6)2+ 769.3 769.3

8 TVEGAGSIAAATGFVKK 81 - 97 a) (536.4)3+ 1606.2 1606.8

9 TKEQVTNVGGAVVTGVTAVAQK 59 - 80 a) (720.1)3+ 2157.2 2157.4

10 TVEGAGSIAAATGFVK 81 - 96 a) (740.2)2+ 1478.5 1478.6

11 EQVTNVGGAVVTGVTAVAQK 61 - 80 a) (643.6)3+ 1927.9 1928.1

12 TKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGK

59 -102 b) (4288.7)1+ 4287.7 4287.8

13 KDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA

97 -140 b) (4959.1)1+ 4958.1 4958.1

9

Supporting Figures

629.723+

658.422+

689.721+

724.120+

762.119+

804.418+

851.617+

904.716+

965.015+

600 650 700 750 800 850 900 950 1000 1050 1100 m/z

629.723+

658.422+

689.721+

724.120+

762.119+

804.418+

851.617+

904.716+

965.015+

600 650 700 750 800 850 900 950 1000 1050 1100 m/z

Mw exp : 14460.3 DaMw calc : 14460.1 DaΔm = 13.83 ppm

20 30 40 500

10

20

30

40

Ab

sorb

ance

(22

0 n

m)

Time (min)

29.8

20 30 40 500

10

20

30

40

Ab

sorb

ance

(22

0 n

m)

Time (min)

29.8

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH

GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL

GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

1

51

101 140

A) B)

Figure S1: HPLC Purification (a) and ESI- mass spectrum (b) of freshly prepared recombinant E. Coli αSynuclein. See

Materials and Methods for HPLC and MS analysis.

807.3

857.6

914.8

980.0

1033.7

1055.3

1113.1

1143.1

1205.8

1247.0

1315.31371.6

1446.81523.9

1607.3

800 900 1000 1100 1200 1300 1400 1500 1600 m/z

11+

12+

13+

17+

16+

15+

14+

14+

13+

12+

11+

964.815+

14+904.6

851.517+

10+

9+10+

9+

1 (1-140)Mw exp : 14459.5 DaMw calc : 14460.1 Da

2 (7-140) Mw exp : 13707.6 DaMw calc : 13708.2 Da

1 (1-140)2 (7-140)

Figure S2: ESI- MS identification of N-terminal truncation product, αSyn(7 – 140), following 3 hrs incubation of αSyn in

PBS buffer, pH 7.5. Edman sequence determination was performed with a Applied Biosystems Procise-470

sequencer as described)[22b].

10

TOFOrthogonal quadrupole

TrapIon mobilityseparation

Transfer

TOFOrthogonal quadrupole

TrapIon mobilityseparation

Transfer

Retention time (mins)

Drift time (bins)

Peak 1

Peak 2

m/z

%

A)

B)

C)

Figure S3. Scheme of the SYNAPT- ion mobility cell between the quadrupole analyzer and the orthogonal TOF analyzer[18a] (a); separation of peaks 1 and 2 (b); ion mobility drift time profile vs. m/z values of the IMS-MS analysis of the in vitro

αSyn incubation mixture for 7 days (c).

Mw exp : 7274.9 DaMw calc : 7274.4 DaΔm = 55 ppm

809.3

910.3

1040.2

600 800 1000 1200 1400 m/z

1213.4728.6

8+

7+

6+

9+

10+

809.3

910.3

1040.2

600 800 1000 1200 1400 m/z

1213.4728.6

8+

7+

6+

9+

10+

30.9

20 30 40 50

0

50

100

150

200

250

300

30.9

20 30 40 50

0

50

100

150

200

250

300

20 30 40 50

0

50

100

150

200

250

300

Ab

sorb

ance

(22

0 n

m)

Time (min)

72TGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA140

A) B)

Figure S4: HPLC Purification (a) and ESI-mass spectrometric characterization (b) of recombinant αSyn(72-140)

fragment expressed in E. Coli.

11

M S5succin(7d)

S5(7d)

400 600 800 1000 1200 1400 1600 1800 m/z

17+16+

15+

14+

13+

18+

19+

20+

Mw exp : 16159.4 DaMw calc : 16161.4 Da

40 -50 -

30 -

25 -20 -

15 -

kDa

10 -

60 -

85 -

200 -

A) B)

Figure S5: Gel electrophoretic and ESI-MS characterization of amino- succinylated αSyn 1.