Figure 3

(a) SDS-PAGE analysis of α-syn samples. 100 μM of purified recombinant human α-syn was oxidized using Cu²⁺/H₂O₂ in 20 mM HEPES buffer, pH 7.4, at 37 °C with agitation of 400 rpm for 5 hours and EDTA was added to quench the oxidation process. SDS-sample buffer was added to the samples and then boiled for 5 min at 100 °C, and 10 μl of the boiled sample loaded on 12% Tris-glycine gel and then silver stained. The figure shows the appearance of a band with molecular weight about 35 kDa after 5 h oxidation indicating the presence of a dimer, which is not present in the non-oxidized control. (b) Shows western blot showing the separated pellet and supernatant labeled using the anti-dityrosine primary antibody. The antibody positive bands are highlighted by arrows which are likely to be dimer and tetramer in supernatant and only dimer and fibrillar species in the pellet. NanoLC-MS/MS analysis of tryptic digested α-syn monomer and dimer that were extracted from electrophoresis gel boxed in panel (a), (c) shows analysis from Mascot Engine. Sequence from the monomer band was complete, but two sequences were missing from dimer band in the oxidized sample, TKEGVLYVGSK and GLSKAK (highlighted in red). (d) Mass spectrum of tryptic digested α-syn dimer showing the appearance of a peak at m/z 1179.6127 that corresponds to the dimer of the peptide with sequence of (₃₃TKEGVLYVGSK₄₃) containing Y39.