Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to alpha-synuclein (alpha-Syn). Three different alpha-Syn species phosphorylated at Serine 87 (S87p-alpha-Syn), Serine 129 (S129p-alpha-Syn) and Serine 87/129 (S87p,129p-alpha-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated alpha-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu2+ ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu2+ were similar between phosphorylated and non-phosphorylated alpha-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated alpha-Syn (Np-alpha-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-alpha-Syn and S87p,129p-alpha-Syn oligomers induced by DA/Cu2+, showed higher toxicity than did S129p-alpha-Syn. When alpha-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu2+ to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. alpha-Syn co-incubated with DA or DA/Cu2+ showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-alpha-Syn. This study supports that phosphorylated oligomers of alpha-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for alpha-Syn aggregation. (C) 2013 Elsevier Inc. All rights reserved.