Identification of Alpha-Synuclein Interacting Synaptosomal Proteins

α-Synuclein is an abundant presynaptic protein that when aggregated and dyslocated from the synapse is associated to Parkinson’s disease and dementia. The normal presynaptic function of α-synuclein is unclear as well as the disease triggering mechanism. In order to extend the knowledge of the α-synuclein interactome during normal function and disease, we have used porcine brain synaptosomes as a source of ligands and purified α-synuclein monomers or oligomers as bait in co-immunoprecipitation experiments. The isolated binding synaptosomal proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using the freely-available Windows client application, Skyline Targeted Proteomic Environment. To compare proteins binding to a-synuclein monomer with proteins binding to a-synuclein oligomer, quantifications were log transformed, normalized to buffer-background, and compared by students t-test. Furthermore, to specify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. 10 α-synuclein preferential monomer binding proteins were identified and among those, we successfully validated Abl interactor 1, and myelin proteolipid protein. 76 α-synuclein preferential oligomer binding proteins were found, including glutamate decarboxylase 2, synapsin 1, and glial fibrillary acidic protein, which were positively validated. We identified 92 proteins binding to α-synuclein, for which we were not able to detect any conformational preferences among. This study presents a catalog of proteins interacting with α-synuclein in its non-aggregated and aggregated oligomeric state, which can be used to investigate the normal and pathological roles of α-synuclein.