Identification of post-translationally modified α-Synuclein protein in biofluids of Parkinson's disease patients using a targeted and quantitative mass spectrometry approach. Céline Vocat1, Bruno Fauvet1, Michel Prudent4, Adrien W. Schmid3, Hilal A. Lashuel1&2. 1 Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. 2. Qatar Biomedical Research Institute, 5825 Doha, Qatar. 3. Proteomics Core Facility, Ecole Polytechnique Fédérale Lausanne (EPFL), Switzerland. 4. Service Régional Vaudois de Transfusion Sanguine, Unité de Recherche et Développement, Switzerland. Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopaminergic neurons and the presence of intracellular protein inclusions (Lewy Bodies) found in the brain of affected patients. Protein aggregation and post-translational modifications (PTMs), such as the site specific phosphorylation of alpha-Synuclein (α-Syn) protein have been reported to be strongly linked to PD pathogenesis. Therefore, pathologically modified α-Syn species represent a primary target for the diagnosis and treatment of PD. In this work, we aimed at conducting a comprehensive study, using multiple mass spectrometry and proteomics based approaches, to assess the chemical heterogeneity of α-Syn and to identify and map the pattern of α-Syn PTMs in plasma and red blood cells from PD and dementia with Lewy bodies (DLB) patients compared to healthy, age-matched control subjects. More specifically, we focused on the pattern of PTMs in the blood in order to identify if these modifications correlate with α-Syn PTM's observed in the brain and cerebrospinal fluid (CSF) during disease progression. The use of full-length, heavy isotope-labelled (15N) α-Syn protein and peptide standards with site-specific modifications, which mirror the key pathological PTMs of α-Syn found in PD, with targeted proteomics and selected reaction monitoring (SRM) mass spectrometry have enabled us to specifically identify and monitor single or multiple site-specific phosphorylations, N-terminal acetylation, truncations and splice variants of α-Syn. We have developed a multiplexed SRM assay which allows us to monitor several PTMs during a single analytical run. The identification of a specific isoform or PTMs pattern that correlate with PD or DLB could provide novel insights into the mechanism of the disease development, contribute to the identification of novel therapeutic targets and most importantly, could provide a diagnostic marker to detect and monitor the progression of PD and related synucleinopathies.


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