Mass Spectrometric Analysis of Lewy Body-Enriched α-Synuclein in Parkinson’s Disease

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Abstract
Parkinson’s disease (PD) is characterized by intra-neuronal inclusions of aggregated α-synuclein protein (so called Lewy bodies) in distinct brain regions. Multiple posttranslational modifications may affect the structure and function of α-synuclein. Mass spectrometry-based analysis may be useful for the characterization and quantitation of α-synuclein forms, but has proven challenging, mainly due to the insolubility of Lewy bodies in aqueous buffer. In the present study, we developed a novel method by combining differential solubilization with immunoprecipitation and targeted proteomics using liquid chromatography and tandem mass spectrometry. Brain tissue homogenization and sample preparation were modified to facilitate analysis of soluble, detergent soluble and detergent insoluble protein fractions (Lewy body-enriched). The method was used to compare α-synuclein forms from cingulate cortex (affected) and occipital cortex (unaffected) in two study sets of PD patients, and controls. We identified ~20 modified α-synuclein variants, including species with N-terminal acetylation and C-terminal truncations at amino acids 103 and 119. The levels of α-synuclein forms Ac-α-syn1-6, α-syn13-21, α-syn35-43, α-syn46-58, α-syn61-80 and α-syn81-96 except Ac-α-syn103-119 were significantly increased in PD cingulate region compared to controls in the Lewy body-enriched α-synuclein fraction. In the soluble fraction, only Ac-α-syn1-6 was significantly increased in PD compared to controls. None of the detected α-synuclein variants was Lewy body-specific, but acetylated forms should be examined further as potential biomarkers for abnormal α-synuclein accumulation.
Funding Information
  • Vetenskapsr?det
  • Parkinson Research Foundation
  • Michael J. Fox Foundation for Parkinson's Research
  • Sahlgrenska Universitetssjukhuset
  • H2020 European Research Council

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