Heat-mediated enrichment of α-synuclein from cells and tissue for assessing post-translational modifications

Authors

  • Hugo Vicente Miranda,

    1. Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal
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  • Wei Xiang,

    1. Institut für Biochemie (Emil-Fischer-Zentrum), Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany
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  • Rita M. de Oliveira,

    1. Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal
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  • Tânia Simões,

    1. Laboratório de Proteómica, Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal
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  • José Pimentel,

    1. Laboratory of Neuropathology, Department of Neurosciences, Serviço de Neurologia, CHLN EPE-Hospital de Santa Maria, Lisboa, Portugal
    2. Neurological Clinical Research Unit, Instituto de Medicina Molecular, Lisboa, Portugal
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  • Jochen Klucken,

    1. Department of Molecular Neurology, University Hospital Erlangen, Erlangen, Germany
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  • Deborah Penque,

    1. Laboratório de Proteómica, Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal
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  • Tiago F. Outeiro

    Corresponding author
    1. Instituto de Fisiologia, Faculdade de Medicina de Lisboa, Lisboa, Portugal
    2. Department of Neurodegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), University Medical Center Göttingen, Göttingen, Germany
    • Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal
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Address correspondence and reprint requests to Dr. Tiago Fleming Outeiro, Department of Neurodegeneration and Restorative Research, University Medical Center Goettingen, Waldweg 33, 37073 Goettingen, Germany. E-mail: touteiro@gmail.com

Abstract

α-synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies.

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In this study, we describe a method for enriching alpha-synuclein (α-syn) from a variety of biological samples, from cultured cells to brain tissues. Enrichment of α-syn was achieved by heating samples, further facilitating the identification of specific post-translational modifications by immunoblot, or mass spectrometry-based techniques. This approach will contribute to the clarification of the role of α-syn PTMs in Parkinson's disease.

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