Identification of full-length and proteolytic fragments of α- α-synuclein

The characterization of the truncated products of αSyn, being unsuccessful using ESI-MS and HPLC-MS, was obtained by ion-mobility mass spectrometry (IMS-MS) consisting of a drift region (Triwave). The Triwave system is composed of three T-Wave devices and is illustrated in Figure 33a.

The DriftScope was used to extract the drift time distributions, which revealed separation of two components (Figure 33b). Generally, the speed with which an ion traverses the drift region depends on its collisional cross section CCS (gas phase size); ions with larger CCSs proceed more slowly than ions with smaller ones. Peak 1 from Figure 33b contained ions with small CCSs and they have an

arrival time distribution shorter than ions from peak 2. Figure 33c shows the HDMS analysis (drift time vs. m/z) of αSyn. The intensity of an ion is indicated using a color scale from least (blue/red) to most (yellow/white) intense.

Orthogonal TOF quadrupole

Trap Ion mobility

separation Transfer

Peak 1

Peak 2

m/z

b

c

%

a

Figure 33: (a), Scheme of Ion-mobility MS system, which incorporates Triwave technology for ion-mobility separations at the limits of MS detection in the SYNAPT HDMS system; (b), Ion-mobility arrival time distribution for ions observed; (c), Plot of m/z versus drift time of αSyn illustrating the multidimensional nature of the data produced by ion-mobility mass spectrometry.

An ion-mobility plot of the αSyn incubation mixture after 48 hours (sample S2;

see Figure 27) provided two peaks with different drift times for the multiply charged ion series, which were analyzed by molecular mass determination and by tandem-MS sequencing (Figure 34). Deconvolution of the multiply charged ion series to singly charged ions provided identifications of intact, full-length αSyn monomer (14459.4 Da) and dimer (28919.6 Da) in peak 2, in agreement with MS data of the gel electrophoretic bands (Figure 27 and Table 9). A most remarkable fragment was identified in the ion series of peak 1 by cleavage between residues Val-71 and Thr-72 at the central amyloidogenic domain (61-95), corresponding to the gel electrophoretic band 4 of ca. 8 kDa (Figure 27).

This fragment was identified as the C-terminal polypeptide αSyn (72-140) by

exact molecular mass determination (7271.1 Da) and tandem-MS sequencing of the 1455.6 (+5) m/z precursor ion (Figure 34b, d).

3906_T002.raw : 1

7250 7260 7270 7280 7290 7300 mass

%

0 100

7250 7260 7270 7280 7290 7300 mass

7271.1191

10000 14000 18000 22000 26000 mass

% 10000 14000 18000 22000 26000 mass

%

Figure 34: Identification of proteolytic truncation products of αSyn by ion-mobility mass spectrometry: (a), HDMS analysis (driftscope data view) of S2 oligomerization-aggregation products of αSyn incubated in PBS for 2 d at 37°C. Signals corresponding to monomers, dimers and truncated peptide sequences are observed. Each pixel represents one ion with color representing intensity from low (blue) to high (yellow); (b), Extracted deconvoluted spectrum of peak 1 is the truncated peptide sequence αSyn of 7.2 kDa (72-140); (c), Extracted deconvoluted spectrum of peak 2 showing monomer plus dimer of αSyn; (d) MS/MS fragmentation of 1455.6 (+5) m/z precursor ion using elevated collision energy in transfer region of Triwave leads to identification of proteolytic truncation product αSyn 72-140.

Figure 35 compares the ion-mobility- MS drift time profiles (driftscope data view) of a freshly prepared αSyn solution, and αSyn- oligomers in incubation mixtures type S5, S6 and S5 + S6 after 7 days of incubation in PBS with 20% ethanol, 10 µM FeCl3. In the different charge state series of ion

signals, αSyn monomer, dimer as well as proteolytic and truncated peptide sequences were identified by molecular weight determinations and partial tandem-MS sequence determinations (Figure 36, Figure 37a and b). The comparison of the extracted drift time “mobilograms” of the ion at m/z 804.1 (18+) between αSyn oligomers type S5, S6, and S5 + S6 is shown in Figure 36.

In the αSyn oligomers type S5, two drift time (conformational) states are present, while in type S5 and S5 + S6 even three drift time states are observed indicating three different structural types.

3282_D023.raw : 1

Figure 35: Ion-mobility drift time of αSyn after 7 days of incubation in 50 mM sodium phosphate buffer, containing 20% ethanol to a final concentration of 7 µM (S5); αSyn after 7 days of incubation in 50 mM sodium phosphate buffer, containing 20% ethanol, 10 µM FeCl₃ (S6); freshly prepared αSyn. Signals corresponding to monomers, dimers and truncated peptide sequences are observed.

sample alphasyn

3282_D025_dt_01 TOF MS ES+

804.074_804.52 0.10Da 937 762.16.30

3282_D024_dt_01 TOF MS ES+

804.164_804.602 0.10Da 440 6.66

804.3

3282_D023_dt_01 TOF MS ES+

804.106_804.602 0.10Da 901 6.39

804.3

3282_D022_dt_01 TOF MS ES+

804.106_804.52 0.10Da

Figure 36: Comparison of the extracted mobilograms of the ion m/z 804.1 between αSyn oligomers type S5, S6, S5 + S6. In αSyn oligomers type S5 two structures are observed; in type S6 and S5 + S6 three main forms are present.

The extracted, deconvoluted mass spectra of the driftscope profile due to the αSyn oligomers, type S5 revealed the αSyn monomer and the intact full-length dimer (Figure 37a; Mw 14459.4; 28919.6 Da), while the oligomer peak, type S5 + S6 showed an additional truncated polypeptide, αSyn (40-140) (Figure 37b;

Mw 10437 Da). Further peptide fragments of αSyn with high aggregating propensity, corresponding to the bands observed by gel electrophoresis (Figure 27) are summarized in Table 9.

14459.4

10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 mass

%

0 100

28919.6

mass 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 mass

% 10000 11000 12000 13000 14000 15000 16000 17000 18000 19000 mass

%

Figure 37: Extracted deconvoluted spectra of αSyn oligomers: (a), Type S5 oligomers are showing αSyn monomer plus dimer; (b), type S5 + S6 oligomers are containing truncated polypeptide sequence at 10.5 kDa (αSyn (40-140)).

In conclusion IMS-MS of the in vitro aggregation of wt-αSyn enabled the structure elucidation of several hitherto unknown truncation and degradation products that are summarized in Table 9 and a proteolytic fragment at V71-T72 in the aggregation domain that appears to be a key intermediate in the aggregation pathway (see 2.7.3).

Table 9: αSyn aggregation products observed by gel electrophoresis and identified by IMS-MS.

αSyn ^a Gel electrophoresis ^b IMS-MS solvent time gel spot Mw gel Mcalc Mexp sequence S1 water 0 d 1 17 kDa 14460.1 14460.0 1-140 S2 20 mM PBS 2 d 4 < 10 kDa 7270.3 7270.1 72-140 S3 20 mM NH4(Ac) 2 d 2 14 kDa 12161.5 12163.0 14-133 S4 20 mM NH4HCO3 3 h 1a 17 kDa 13708.2 13706.6 7-140 S4 20 mM NH4HCO3 2 d 4 < 10 kDa 7270.3 7270.1 72-140 S5 PBS 7 d 3 10 kDa 10436.4 10436.0 40-140 S6 PBS 7 d 3 10 kDa 10436.4 10436.0 40-140

a αSyn aggregation has been studied under various conditions which differ in used buffer systems and incubation time

b Aggregation products of αSyn were observed by Tris-tricine gel electrophoresis

These results, showing IMS-MS as a powerful tool for the analysis of reactive intermediates due to its “affinity”-like separation ability, of a protein aggregation pathway suggest proteolytic fragments as a key molecular species involved in the “misfolding” and aggregation of αSyn.

2.4 Epitope identification of synuclein- antibodies by affinity-mass