characterization of oligomerization–aggregation products of neurodegenerative target proteins by...

7/28/2019 Characterization of OligomerizationAggregation Products of Neurodegenerative Target Proteins by Ion Mobility Ma

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Chapter 27

Characterization of OligomerizationAggregation Productsof Neurodegenerative Target Proteins by Ion Mobility MassSpectrometry

Camelia Vlad, Marius Ionut Iurascu, Stefan Slamnoiu,Bastian Hengerer, and Michael Przybylski

Abstract

Protein amyloidogenesis is generally considered to be a major cause of two most severe neurodegenerativedisorders, Parkinsons disease (PD) and Alzheimers disease (AD). Formation and accumulation of fibrillaraggregates and plaques derived from a-synuclein (a-Syn) and -amyloid (A) polypeptide in brain havebeen recognized as characteristics of Parkinsons disease and Alzheimers disease. Oligomeric aggregates ofa-Syn and A are considered as neurotoxic intermediate products leading to progressive neurodegenera-tion. However, molecular details of the oligomerization and aggregation pathway(s) and the molecularstructure details are still unclear. We describe here the application of ion-mobility mass spectrometry (IMS-MS) to the identification ofa-Syn and A oligomerizationaggregation products, and to the characteriza-

tion of different conformational forms. IMS-MS is an analytical technique capable of separating gaseousions based on their size, shape, and topography. IMS-MS studies of soluble a-Syn and A-aggregatesprepared by in vitro incubation over several days were performed on a quadrupole time of flight massspectrometer equipped with a travelling wave ion mobility cell, and revealed the presence of differentconformational states and, remarkably, truncation and proteolytic products of high aggregating reactivity.These results suggest that different polypeptide sequences may contribute to the formation of oligomericaggregates of heterogeneous composition and distinct biochemical properties.

Key words: Parkinsons disease, a-Synuclein, Alzheimers disease, -Amyloid, Oligomerization, Ionmobility mass spectrometry

1. Introduction

Soluble amyloid oligomers have been generally thought to beformed as common intermediates in the amyloid fibrillization path-

way, and have been implicated as neurotoxic species of amyloid-related neurodegenerative diseases, such as Alzheimers disease(AD) and Parkinsons Disease (PD). In the last few years, increasing

Vladimir N. Uversky and A. Keith Dunker (eds.), Intrinsically Disordered Protein Analysis:Volume 2, Methods and Experimental Tools, Methods in Molecular Biology, vol. 896,DOI 10.1007/978-1-4614-3704-8_27,# Springer Science+Business Media New York 2012

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evidence has been obtained that soluble oligomers, and not highmolecular weight fibrillar end products, are highly toxic, even whenthey are formed from proteins that are not normally related toneurodegenerative processes (1, 2). Formation and accumulationof fibrillar plaques and misfolding- aggregation products of

-amyloid peptide (A) and a-synuclein (a-Syn) in brain havebeen recognized as characteristics of Alzheimers disease (AD)and Parkinsons disease (PD) (36). Moreover, in recent years anincreasing number of proteins have been identified as being intrin-sically disordered (Intrinsically Disordered Proteins; IDPs), sug-gesting that they lack stable, folded tertiary structures underphysiological conditions (7). A number of intrinsically disorderedproteins have been shown, or are suspected to be associated

with human diseases as cancer, cardiovascular disease, amyloidoses,neurodegenerative diseases, and diabetes. IDPs, such as a-synuclein

involved in Parkinsons disease (8, 9), protein-Tau in Alzheimersdisease (AD) and related tauopathies (10), and prion protein (PrP)in prion disorders (PrD) such as CreutzfeldtJakob Disease (CJD),GerstmannStrausslerScheinker Syndrome and Fatal FamilialInsomnia (FFI) (11, 12) represent potentially crucial targets fordrugs that modulate their proteinprotein interactions.

a-Synuclein, a protein of 140 amino acids, is mainly expressedin the presynaptic terminals of neurons and has been stronglycorrelated with Parkinsons disease, one of the most severe neuro-degenerative motor disorders (8, 9). a-Syn is a natively unfolded

protein with unknown function and unspecific conformational het-erogeneity and properties. The main evidence for a causal role ofa-Syn in PD came from the discovery of three rare point mutations ofthe amino acid sequence, A53T (13), A30P (14), and E46K (15) infamilies with autosomal dominant Parkinsons Disease that lead toa-Syn accumulation in Lewy bodies and other pathological inclu-sions at conditions inducing PD (16).

-Amyloid (A) is a neurotoxic polypeptide containing 3943amino acid residues and is derived from proteolytic cleavage of thetransmembrane A precursor protein (APP). Recently, the forma-

tion of A-oligomers has become of particular interest, since oligo-meric aggregates have been suggested to be key neurotoxic speciesfor progressive neurodegeneration (3, 17, 18); however, moleculardetails of the pathophysiological degradation of APP, and of the

A-aggregation pathways and structures are hitherto unclear (19).In recent years, mass spectrometry (MS) has become a major

analytical tool in structural biology (20). In particular, electrospraymass spectrometry (ESI-MS) has emerged as a powerful techniquefor producing intact gas- phase ions from large biomolecular ions,and ions due to supramolecular complexes (21). However, in con-

trast to the large number of ESI- mass spectrometric studies ofprotein structures, structure modifications and applications to pro-teomics (2228), the molecular characterization of protein

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misfolding and aggregation species by mass spectrometry hadlittle success; possible explanations are (1) the low intermediateconcentrations and (2) and slow rates of aggregate formationin vitro (6, 7). Conventional soft-ionization mass spectrometrymethods such as ESI-MS and HPLC-MS are not suitable to direct

in-situ analysis of conformational states and intermediates atdifferent concentrations. Recently, ion mobility mass spectrometry(IMS-MS) is emerging as a new tool to probe complex biomolecu-lar structures from solution phase structures, due to the potential ofIMS-MS for separation of mixtures of protein complexes by con-formational state, and spatial shape and topology (2936). TheIMS-MS instrument employed in this study consists of two parts:(1) an ion mobility drift cell where ions are separated within anelectric field according to their collisional cross sections and (2) aquadrupole time-of-flight mass spectrometer (Synapt-QTOF-MS)

(33, 34, 3638). Thus, IMS-MS implements a new mode of sepa-ration that allows the differentiation of protein conformationalstates. In the present study, ESI-MS (21) and IMS-MS have beenapplied to the direct analysis of mixtures of a-Syn and A (38)aggregation products in vitro, formed during prolonged incuba-tion times.

2. Materials

2.1. Preparation

and Characterization

ofa-Synuclein and

A(140) Oligomers

Alpha-Syn and A (140) oligomers were prepared by an in vitroincubation procedure for up to 7 days at 37C and at 20C (38, 39)(see Note 2). a-Syn oligomers type A1 were prepared by dissolvingthe protein in 50 mM sodium phosphate buffer (pH 7.0) contain-ing 20 % ethanol, to a final concentration of 7 mM (39). In the caseof oligomers type A2, 10 mM FeCl3 was added, while oligomerstype A1 were prepared without adding FeCl3. After 4 h of shaking,both types of oligomers were lyophilized and resuspended in50 mM sodium phosphate buffer (pH 7.0) containing 10 % ethanol

with a half of the starting volume. This was followed by incubationfor 7 days at room temperature For A-oligomerization, A(140)was solubilized at a concentration of 1 mg/mL in a buffer containing50 mM Na3PO4, 150 mM NaCl, 0.02 % NaN3 at pH 7.5 (38).a-Syn and A(140) oligomers were separated by gel electrophore-sis and subjected to IMS-MS measurements.

2.2. Gel Electrophoresis

ofa-Synuclein and

A(140) Oligomers

and Aggregates

Separation of a-Syn and A (140) oligomers was performed byone-dimensional Tristricine polyacrylamide gel electrophoresis(Tristricine PAGE) on a Mini-Protean II or Mini-Protean 3 elec-

trophoresis cell (BioRad, M

unchen, Germany) using 12 % and 15 %polyacrylamide gel electrophoresis, and protein bands visualizedby Coomassie Blue staining. The dimensions of the gel were

27 Characterization of OligomerizationAggregation Products of Neurodegenerative. . . 401


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90 60 1 mm. The proteins were solubilized and denaturedusing a stock solution of the twofold concentrated sample buffercontaining 4 % SDS, 25 % Glycerin, 50 mM TrisHCl, 0.02 %Coomassie, 6 M urea, pH 6.8. Gels were run at 60 V until thetracking dye entered the separating gel and at 120 V until the

tracking dye reached the bottom of the gel.

2.3. Buffers and Stock

Solutions

The following solutions were prepared in ultrapure water (preparedby purifying deionized water to attain a conductivity of 18 MO):

1. a-Syn oligomers type A1 buffer: 50 mM sodium phosphatebuffer, pH 7.0. Weigh 1.779 g Na2HPO4 and transfer to a200-ml graduated glass beaker. Add ultrapure water to a

volume of 200 ml. Mix and adjust pH with HCl (see Note3). Add 40 ml ethanol. Store at 4C.

2. a-Syn oligomers type A2 buffer: 50 mM sodium phosphatebuffer, pH 7.0. Weigh 1.779 g Na2HPO4 and transfer to aglass beaker. Add ultrapure water to a volume of 200 ml. Mixand adjust pH with HCl (see Note 3). Add 40 ml ethanol and0.3244 mg FeCl3. Store at 4

C.

3. A(140) oligomers buffer: 50 mM Na3PO4, 150 mM NaCl,0.02 % NaN3 at pH 7.5. Weigh 1.9 g Na3PO4 and 0.88 gNaCl and transfer to a 100-ml graduated glass beaker contain-ing about 80 ml ultrapure water. Mix and adjust pH with HCl(see Note 3). Make up to 100 ml with ultrapure water. Store

at 4C.

For Tristricine PAGE, the following solutions were preparedin ultrapure water.

4. TrisHCl/SDS buffer: 3 M Tris, 0.3 % SDS, pH 8.45. Weight36.4 g Tris and transfer to a 100 ml graduated glass beakercontaining about 60 ml ultrapure water. Mix and adjust pH

with HCl to 8.45 (see Note 3). Add 0.3 g SDS and make up to100 ml with ultrapure water.

5. 2 Tricine sample buffer: 100 mM Tris pH 6.8, 24 % Glyc-

erol, 8 % SDS, 0.02 % Coomassie brilliant blue G-250. Weigh1.21 g Tris and transfer to a 10-ml graduated glass beaker.

Adjust pH with HCl to 6.8 (see Note 3). Add 2.4 ml Glycerol,0.8 g SDS (see Note 4), and 2 mg Coomassie brilliant blue G-250. Store at 4C.

6. 1 Cathode buffer (upper chamber): 100 mM Tris, 100 mMTricine, 0.1 % SDS. Weigh 6.055 g Tris, 8.96 g Tricine, 0.5 gSDS and transfer to a 500-ml graduated glass beaker andmake up with ultrapure water.

7. 1 Anode buffer (lower chamber): 20 mM Tris, pH 8.96.Weigh 12.11 g Tris and transfer to a 500-ml graduated glassbeaker containing about 400 ml ultrapure water. Mix and

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adjust pH with HCl to 8.96 (see Note 3). Make up withultrapure water to 500 ml.

8. Ammonium persulfate (APS): 10 % solution in water (seeNote 5).

9. N0

,N0

,N0

,N0

-tetramethyl-ethylenediamine (TEMED).10. 15 % separating gel: Mix 7.5 ml Acrylamide, 5 ml TrisHCl/

SDS and 0.9 ml ultrapure water in a glass beaker. Add 1.58 mlGlycerol and sonicate until total solubilization. Add 75 ml

APS, 10 ml TEMED and pour the gel into casting platesleaving room at the top for the stacking gel and comb. Addultrapure water to cover top of gel and make flat interface.Polymerize for about 30 min. Check that remaining gel inglass beaker is polymerized before proceeding.

11. Stacking gel: Mix in a glass beaker 972 ml Acrylamide,

1.86 ml TrisHCl/SDS, 4.67 ml ultrapure water, 40 mlAPS and 7.5 ml TEMED and pour gel on top of alreadypolymerized separating gel. Insert comb and allow to poly-merize (about 30 min).

3. Methods

3.1. Gel ElectrophoreticIsolation and Primary

Structure

Characterization

ofa-Synuclein

Oligomerization

Aggregation

Products

The in vitro oligomerization products of a-Syn were firstcharacterized by Tristricine PAGE, as described in Subheadings 2.1and 2.2, and the isolated protein bands analyzed by mass spectrom-etry. The electrophoretic separation revealed bands with molecular

weights corresponding to monomeric (a-Syn)1, dimeric (a-Syn)2,oligomeric (a-Syn)

nproteins, and three additional bands below the

a-Syn monomer that were assigned to truncated forms ofa-Syn (seeFig. 1). The band in the region of approximately 37 kDa was excisedfrom the gel, digested with trypsin, and analyzed by HPLC-MS withan ESI-ion trap mass spectrometer (see Note 6). The mass spectro-

metric data ascertained the presence ofa-Syn peptides by identifica-tion of the corresponding peptide fragments, andidentified ana-Syndimer composed of two intact a-Syn polypeptides. This result wasascertained by N-terminal Edman sequence determination (seeFig. 1c) (see Note 7). Following electrophoretic separation, thebands were transferred onto a PVDF membrane and subjected toEdman sequencing which provided the identification of N-terminaltruncation of the band at 13.9 kDa, a-Syn(5140). Direct massspectrometric analysis of monomer (a-Syn)1 was performed withan Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics,

Bremen, Germany). The protein band was solubilized in 1 % formicacid as a volatile solvent, to a final concentration of 30 mM, pH 2.5.



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The spectrum was obtained in the positive ion mode (see Fig. 1b).Inthe ESI mass spectrum, a series of multiply charged ions wereobtained with the most abundant ion at charge state +19.

3.2. Ion-Mobility Mass

Spectrometry

Ion-mobility mass spectrometry was performed with a SYNAPT-QTOF-MS (Waters, Manchester, UK) equipped with an nano-electrospray ionization source (2225). The ions were passedthrough a quadrupole and either set to transmit a substantial massrange or to select a particular m/z before entering the Triwave ionmobility cell. The Triwave system (22) is composed of three

T-Wave devices (Waters SYNAPT). The first device, theTrap T-Wave, accumulates ions which are stored and gated(500 msec) into the second device, the ion mobility T-Wave cell,

where they are separated according to their cross section-dependent mobilities (22, 23). The final transfer T-Wave is usedto transport the separated ions into the orthogonal acceleration oa-TOF for MS analysis. The injection volume was 5 ml, and a priordesalting step was performed on a cartridge for 10 min at 20 ml/min with gradient of 1090 % acetonitrile. The mass spectrometricacquisition characteristics were: 3504,000 m/z, cone voltage

25 V, IMS pressure 0.45 bar, wave height 515 V. IMS-MS dataare inherently three-dimensional, consisting of mass, drift time andintensity (relative abundance) for all ions observed.

Fig. 1. (a), Primary structure of a-synuclein (a-Syn). (b), TrisTricine PAGE of a-Syn stained with Coomassie Blue:

molecular weight marker (lane 1); (A1) a-Syn incubated in PBS buffer at room temperature for 7 days (lane 2); (A2) a-Syn

incubated in PBS buffer with 10 mM FeCl3 at 20C for 7 days (lane 3). (c), Edman sequence determination of (a-Syn)2; ESI-

MS of a-Syn in 1 % aqueous formic acid at a final concentration of 34.5 mM, pH 2.5 showing the 15+ to 23+ charged

molecular ions; Edman sequencing of N-terminal truncated polypeptide sequence of 13.9 kDa, a-Syn(5140).

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3.3. Ion Mobility

Mass Spectrometry

ofa-Syn- Oligomers

Figure 2 summarizes the ion-mobility- MS drift time profiles of afreshly prepared a-Syn solution, and a-Syn- oligomers in incuba-tion mixtures type A1, A2 and A1 + A2 after 7 days of incubation inphosphate-buffered saline with 20 % ethanol, 10 mM FeCl3 (driftscope data view). In the different charge state series of ion signals,a-Syn monomer, dimer as well as proteolytic and truncated peptidesequences were identified by molecular weight determinations andpartial tandem-MS sequence determinations (see Figs. 3 and 4a, b).

The comparison of the extracted drift time mobilograms of theion at m/z 804.1 (18+) between a-Syn oligomers type A1, A2, andA1 + A2 is shown in Fig. 3. In the a-Syn oligomers type A1, twodrift time (conformational) states are present, while in type A2 and

A1 + A2 even three drift time states are observed indicating threedifferent structural types. The extracted, deconvoluted mass spectraof the drift scope profile due to the a-Syn oligomers, type A1revealed the a-Syn monomer and the intact full-length dimer (seeFig. 4a; m.w. 14,459; 29.919 Da), while the oligomer peak, type

A1 + A2 showed an additional truncated polypeptide, a-Syn

(40140) (see Fig. 4b; m.w. 10.437 Da); further proteolyticpeptide fragments of a-Syn with high aggregating reactivity,corresponding to the proteolytic bands observed by gel electropho-resis (see Fig. 1) have been recently identified (40).

Fig. 2. Ion-mobility drift time of a-Syn after 7 days of incubation in 50 mM sodium phosphate buffer, containing 20 %

ethanol to a final concentration of 7 mM (A1); Syn after 7 days of incubation in 50 mM sodium phosphate buffer, containing

20 % ethanol, 10 mM FeCl3 (A2); freshly prepared a-Syn. Signals corresponding to monomers, dimers, and truncated

peptide sequences are observed.



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3.4. Ion Mobility Mass

Spectrometry

of A-Oligomers

The soluble fraction of the A(140) fibril preparation obtained byincubation over 5 days at 37C, and a freshly prepared A(140)peptide solution (220 mM) were subjected to comparative analysisby ion mobility- MS (see Fig. 5a, b). In the freshly preparedsolution of A(140), the [M+5H]5+ ion was predominant, whilein soluble fibril preparations ions with increasingly higher chargestates ([M+6H]6+, [M+7H]7+) were most abundant, indicative of

oligomer formation. In the gel electrophoretic analysis, the forma-tion of A- oligomers can be directly observed (38). The signal-to-noise ratios of the [M+5H]5+ and [M+6H]6+ ions was found to besignificantly lower in the fibril preparation sample compared to thesample of freshly prepared A(140), suggesting a decreasedamount of A(140) monomer due to the formation of oligomers.The extracted ion mobility profiles for the [M+5H]5+ ion of thefreshly prepared A(140) (see Fig. 5c) compared to that of thefibril preparation (see Fig. 5d) indicate the presence of differentconformational states (A and B) with different ion mobilities (cross

sections), indicative of the oligomerization process. Furthermore,partial oxidation of A at the Met-35 residue has been observed insamples upon formation of oligomers (38).

sample alphasyn

Scan

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165

%

0

100

3282_D025_dt_01 TOF MS ES+804.074_804.52 0.10Da

937

6.30762.1

3282_D024_dt_01 TOF MS ES+804.164_804.602 0.10Da

440

6.66804.3

3282_D023_dt_01 TOF MS ES+804.106_804.602 0.10Da

901

6.39804.3

3282_D022_dt_01 TOF MS ES+804.106_804.52 0.10Da

4.26e3

6.12;851.5

A1 +A2(7 days)

A2

(7 days)

a

b

c

d

A1

(7 days)

-Syn(0 days)

Fig. 3. Comparison of the extracted mobilograms of the ion m/z 804.1 between a-Syn oligomers type A1, A2, A1 + A2. In

a-Syn oligomers type A1 two structures are observed; in type A2 and A1 + A2 three main forms are present.

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TOF MS ES+14459.4

mass10000 12000 14000 16000 18000 20000 22000 24000 26000 28000

%

0

1002.30e4

28919.6

-Syn (1-140)

monomer

-Syn (1-140)

dimer

MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV

VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA

1

71 140

-Syn (1-140)

Mw exp : 14459.4 Da

a

Mw calc : 14460.1 Da

m = 48.41 ppm

mass10000 11000 12000 13000 14000 15000 16000 17000 18000 19000

%

0

100

b

TOF MS ES+7.93e3

14461.0010

10437.0010

10457.0010

14477.0010

-Syn (1-140)

-Syn (40-140)

-Syn (1-140)

Mw exp : 14460.0 Da


m = 6.92 ppm

-Syn (40-140)

Mw exp : 10437.0 Da


m = 57.49 ppm

Fig. 4. Extracted deconvoluted spectra of a-Syn oligomers: (a), Type A1 oligomers are showing a-Syn monomer plus

dimer; (b), type A1 + A2 oligomers are containing truncated polypeptide sequence at 10.5 kDa (a-Syn(40140)).



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m/z400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200

%

0

100

%

0

100

3282_D019_dt_01 61 (5.400) Cm (26:104) TOF MS ES+2.02e3866.4890

722.2233

719.2616

702.7539616.7699

616.6233

602.9120

600.3329

619.3473

722.4017

866.2936

722.5703

739.4684

843.4893

739.6289

843.3071

840.8661

739.8396

741.4360

866.6954

866.9017

867.1082

867.31461083.1299

900.78231082.6321

901.0260

924.3208

1083.3848

1083.6399

1083.8828

3282_D018_dt_01 62 (5.490) Cm (30:141)

TOF MS ES+297722.2233

719.5585

703.3997

619.3381

616.7699

612.3430

401.1952610.3362

461.2322

619.4943

722.5703

866.6737

722.7389

866.4781

732.2323

739.6188

866.2610

739.6589

741.4160

866.8800

867.0756

878.2579

878.2799

878.4877

900.4834

900.7712 1083.0813

5+

5+

6+

6+

4+

4+

7+

7+

A

B

a

b

c

d

Scan2 6 2 8 3 0 3 2 3 4 3 6 3 8 4 0 42 4 4 4 6 4 8 5 0 52 5 4 5 6 5 8 6 0 62 6 4 6 6 6 8 7 0 72 74 7 6 7 8 8 0 8 2 8 4 8 6 8 8 9 0 92 9 4 9 6 9 8 100 1 02

%

0

100

26 28 3 0 3 2 3 4 3 6 38 40 4 2 4 4 4 6 4 8 5 0 5 2 5 4 5 6 58 60 6 2 6 4 6 6 6 8 70 7 2 7 4 7 6 7 8 80 8 2 8 4 8 6 8 8 90 92 9 4 9 6 9 8 100 102

%

0

1005+866.6

5+866.6

Drift Time (scans)40 60 80 100

Fig. 5. Ion mobility MS analysis of (a) freshly prepared A(140); (b) supernatant after 5 days incubation; (c) and (d)extracted ion mobility profiles for m/z 866.6, [M+5H]5+ from freshly prepared A(140) and supernatant, respectively.

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4. Notes

Ion mobility mass spectrometry has been shown to be an efficienttool for the characterization of in vitro a-Syn and A oligomeriza-tionaggregation products, and provides evidence of (1), the pres-ence of distinct conformational forms of a-Syn and (2), thepresence of at least two different conformational forms involvedin A- aggregation. These results suggest IMS-MS as a powerfultool for the analysis of reactive intermediates in the aggregation ofintrinsically disordered proteins due to its affinity-like gas phaseseparation capability. In the case ofa-Syn, the IMS-MS data iden-tified proteolytic fragments as key molecular species involved inmisfolding and aggregation pathways, suggesting that the IMS-MS method can also be successfully applied to the identification ofhitherto undetected proteolytic truncation products of neurode-generative target proteins. Thus, gel electrophoresis protein Tau,another key protein in Alzheimers disease shows the formation oftruncation/proteolytic fragments in addition to oligomerizationproducts (see Fig. 6). It will be of interest to systematically exploreIMS-MS in the study of aggregation pathways of intrinsically disor-dered proteins.

1. a-Syn was purified prior to use by analytical RP-HPLC on aUltiMate 3000 system (Dionex, Germering, Germany),

Fig. 6. SDS-PAGE separation of two isoforms of protein-Tau: Molecular weight marker(lane 1); 2 mg Tau2N/4R protein (lane 2) and 2 mg Tau0N/3R protein (lane 3) showing

bands corresponding to monomeric (Tau)1, oligomeric (Tau)nv, and truncated forms of Tau,

isoforms DN(Tau).



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equipped with a Vydac C4 column (250 4.6 mm I.D.) with5 mm silica (300 A pore size) (Hesperia CA). Linear gradientelution (0 min0 % B; 5 min0 % B; 50 min90 % B) witheluent A: (0.1 % trifluoroacetic acid in water) and eluent B:(0.1 % trifluoroacetic acid in acetonitrilewater 80:20 v/v) was

employed at a flow rate of 1 ml/min.

2. A(140) was synthesized by solid phase peptide synthesis on aNovaSyn TGR resin using Fmoc chemistry by a semiautomatedsynthesizer EPS-221 (Abimed, Germany). Fmoc amino acids,NovaSyn TGR resin, PyBop, and other reagents were obtainedfrom Novabiochem (Laufelfingen, Switzerland). The crudeproducts were purified by analytical RP-HPLC (see Note 1).

3. Concentrated HCl (12N) can be used at first to narrow the gapfrom the starting pH to the necessary pH and further use HCl

(1N) until the pH 7 is obtained.4. SDS precipitates at 4C. Therefore, the 2 Tricine sample

buffer needs to be warmed prior to use.

5. The best is to prepare fresh solution each time.

6. Protein spots were excised manually from the gel, cutting asclose to the spots as possible, in order to minimize the contami-nation with SDS. After distaining, the dried gel pieces wereswollen in digestion buffer (12 ng/ml TPCK-trypsin in50 mM NH4HCO3) at 4

C (on ice) for 45 min, and then theywere incubated at 37C overnight (12 h) and at last the peptideswere extracted twice with acetonitrile0.1 % trifluoroacetic acid3:2.

7. The spots of interest were excised, destained with 100 % meth-anol, and applied into the sequencing cartridge. Sequencedeterminations were performed on an Applied BiosystemsModel 494 Procise Sequencer attached to a Model 140 CMicrogradient System, a 785A Programmable AbsorbanceDetector, and a 610A Data Analysis System.

Acknowledgments

We thank Adrian Moise for expert assistance with Edman sequenceand Nick Tomczyk and Emmanuelle Claude, Waters MS- Technol-ogy Centre Manchester, UK, for assistance with the ion mobility-MS. We are grateful to David Clemmer and Nicolas Pearson forhelpful discussion and comments on IMS-MS. This work has been

supported by the Graduate School Chemical Biology, the ResearchCentre Proteostasis, University of Konstanz, and the Landesstif-tung fur Wissenschaft und Forschung Baden- Wurttemberg.

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