Analyses of Detergent-Soluble Oligomers and Mature Aggregates. The soluble oligomers and insoluble aggregates in transfected cells also were examined by exactly following the differential extraction method described in ref. 1. Briefly, differentiated SH-SY5Y cells were scraped onto PBS containing 1% Triton X-100 and protease inhibitors, extracted for 10 min on ice, and centrifuged at 16,000 ´ g for 10 min. The insoluble fraction was resuspended in one-half of initial volume in PBS containing 1% SDS. Equal volumes of both fractions were subjected to immunoblot analysis.

Chromatographic Analyses of a -Syn. For size-exclusion chromatography, a column packed with Saphacryl S-200 HR (Amersham Pharmacia) and equilibrated with PBS (pH 7.4) was used. The PBS-soluble brain homogenates were loaded onto the column, and the proteins were eluted with PBS at 0.8 ml/min. Then, 1.5-ml fractions were collected, and the elution of a -synuclein (a -Syn) was monitored by dot blot and immunoblot analysis.

For anion exchange chromatography, column packed with DEAE Sepharose FF (Amersham Pharmacia) was fully equilibrated with 20 mM Tris•HCl (pH 7.4). Samples were loaded on the column and washed with 20 mM Tris•HCl, and the proteins were eluted with a NaCl linear gradient (buffer A, 20 mM Tris•HCl, pH 7.4; buffer B, 1 M NaCl/20 mM Tris•HCl, pH 7.4) at 3 ml/min. Each fraction collected (3 ml) was monitored for a -Syn by dot blot and immunoblot analysis. Under these conditions, a -Syn12 elutes with » 250 mM NaCl, and a -SynFL elutes with 500 mM NaCl.

Ciphergen Surface-Enhanced Laser Desorption and Ionization (SELDI)-MS Analysis of a -Syn variants. Immunoprecipitated a -Syn variants were eluted with 50% acetonitrile/0.5% trifluoracetic acid (TFA) for mass spectrometry (MS) and directly applied to the Ciphergen H-50 (reverse-phase surface) ProteinChip (Ciphergen, Fremont, CA) for Ciphergen SELDI-TOF MS. After air-drying onto the H-50 ProteinChip, the spot was washed with 0.1% TFA, 0.5 m l of sinapinic acid (saturated solution in 50% acetonitrile/0.1% TFA) matrix was applied on top of the sample, air dried, and analyzed by the Ciphergen Proteinchip System II. The parameters used were as follows: detector sensitivity 10, laser intensity 220, data acquisition method set to SELDI Quantitation, with optimized mass range of 5,000 to 20,000 Da. Calibration standards used were bovine ubiquitin (8,564.800 Da), bovine cytochrome c (12,230.920 Da), and bovine superoxide dismutase 1 (15,591.400 Da).

For the analysis of the C-terminal fragments, a -SynFL and a -Syn12 from human brains were separated by DEAE AEC and immunoprecipitated. The individual a -Syn species were digested with modified sequencing grade trypsin in 25 mM fresh ammonium carbonate (18 h at 37°C). The trypsin digest was concentrated 10-fold by evaporation, mixed 1:1 with the binding buffer (100 mM Tris•HCl, pH 6.5/0.05% Triton X-100), applied (5 m l) to the Ciphergen strong anion exchange chip (SAX2), and incubated for 2 h in humidity chamber. After the incubation, spots were washed (three times each) with the binding buffer and double-distilled H₂O. Following the last wash, 0.5 m l of 1:4 dilution of saturated a -cyano-4-hydroxycinnamic acid (CHCA) was applied to the spot, and the masses of the peptides were analyzed by Ciphergen Protein System II. The parameters used were as follows: detector sensitivity 7, laser intensity 170, and the optimized mass range of 1,000-6,000 Da.

MALDI-TOF MS Analysis of Human (Hu) a -Syn. Immunoprecipitated a -Syn variants were eluted with 50% acetonitrile/0.5% TFA and then dried by SpeedVac concentrator (Savant). The dried protein pellet was digested by sequencing grade trypsin (Roche, Indianapolis). Digested Peptide fragments were desalted by C18 ZipTip (Millipore) and spotted onto the sample plate. The sample then was covered with matrix (10 mg/ml CHCA in 50% acetonitrile/0.3% TFA) and analyzed by MALDI-TOF MS (PerSeptive Biosystems, Framingham, MA). Calibrants were bradykinin fragment 1-7 (757.3997 Da, monoisotopic), human angiotensin II (1,046.5423 Da, monoisotopic), and human ACTH fragment 18-39 (2,465.1989 Da, monoisotopic).

Data were acquired manually, and parameters used were as follows: acquisition mass range (500–5,000 Da), laser intensity (2,200), and operation mode (reflector). Both positive and negative polarities were used to acquire data. The deisotoped spectra, after subtraction of blank spectra, were searched against theoretical trypsin digestion fragments of full-length human a -synuclein (generated by MS-DIGEST, http://prospector.ucsf.edu; maximum of two missed cleavages were allowed), by using MS-BRIDGE (http://prospector.ucsf.edu). The possible truncated forms of a -Syn (Syn1-119, -1-120, -1-121, -1-122, -1-123, and -1-124) also were searched to find the potential C-terminal trypsin digestion fragments.

Cell-Free in Vitro Assembly of a -Syn. Self-assembly of a -Syn was examined by using cell-free in vitro aggregation assay (2) with modifications. Hua -Syn species were expressed in COS-1 cells by transient transfection, and the cells were harvested in PBS (pH 7.2) containing protease inhibitors and homogenized by using a glass-on-glass pestle homogenizer. The homogenate was centrifuged (150,000 ´ g, 45 min at 4°C), and the resulting supernatant (S150) was adjusted to 2 mg/ml of total protein. Concentrations of a -Syn in S150 were determined by quantitative immunoblot analysis (3) against known amounts of purified recombinant GST-a -Syn fusion protein. Desired a -Syn concentrations were obtained by diluting S150 containing a -Syn with the appropriate volumes of S150 from empty pCD vector-transfected COS1 cells. Self-assembly of a -Syn was initiated by incubation at either 37°C or 55°C with shaking. At various times, assembled a -Syn was collected by centrifugation using an airfuge (150,000 ´ g, 10 min at 25°C), and the amounts of a -Syn in the pellet fraction, representing the assembled a -Syn, were determined by the quantitative immunoblot analysis using Syn-1 antibody and the known amounts of recombinant a -Syn standards (3). Consistent with previous in vitro studies, preliminary characterization of the conditions used showed that the assembly plateau is reached by 48–72 h with similar amounts of aggregation between a -Syn species. Thus, the 24-h time point, representing the active aggregation phase, was used to compare the assembly properties of a -Syn species.

1. Lee, H. J., Khoshaghideh, F., Patel, S. & Lee, S. J. (2004) J. Neurosci. 24, 1888–1896.

2. Uversky, V. N., Lee, H. J., Li, J., Fink, A. L. & Lee, S. J. (2001) J. Biol. Chem. 276, 43495–43498.

3. Li, W., Lesuisse, C., Xu, Y., Troncoso, J., Price, D. L. & Lee, M. K. (2004) J. Neurosci. 33, 7400–7409.