Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

Authors

  • Christoph Biesemann,

    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Mads Grønborg,

    1. Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany
    2. Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Current affiliation:
    1. Department of Beta Cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark
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  • Elisa Luquet,

    1. CNRS, IINS, UMR 5297, Bordeaux, France
    2. University of Bordeaux, IINS, UMR 5297, Bordeaux, France
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  • Sven P Wichert,

    1. Gene Expression Group, Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany
    Current affiliation:
    1. Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Munich, Germany
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  • Véronique Bernard,

    1. INSERM U952, Université Pierre et Marie Curie, Paris, France
    2. CNRS UMR 7224, Paris, France
    3. Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France
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  • Simon R Bungers,

    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Ben Cooper,

    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Frédérique Varoqueaux,

    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Liyi Li,

    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Jennifer A Byrne,

    1. Molecular Oncology Laboratory, Children's Cancer Research Unit, Kids Research Institute, Westmead, NSW, Australia
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  • Henning Urlaub,

    1. Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    2. Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany
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  • Olaf Jahn,

    1. Proteomics Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Nils Brose,

    Corresponding author
    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
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  • Etienne Herzog

    Corresponding author
    1. Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany
    2. CNRS, IINS, UMR 5297, Bordeaux, France
    3. University of Bordeaux, IINS, UMR 5297, Bordeaux, France
    4. INSERM U952, Université Pierre et Marie Curie, Paris, France
    5. CNRS UMR 7224, Paris, France
    6. Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France
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Abstract

For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease.

Synopsis

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This study describes the establishment, validation, and application of a new fluorescence-activated sorting method - Fluorescence Activated Synaptosome Sorting or FASS - that allows for purification of glutamatergic synaptosomes from knock-in mutant mice expressing the fluorescent synaptic marker protein VGLUT1-Venus.

  • Fluorescence Activated Synaptosome Sorting from samples of VGLUT1-Venus knock-in mice enriches glutamatergic synaptosomes to near homogeneity.
  • Glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting contain bona fide glutamatergic synapse proteins but lack other synapse types and glia cell contaminants.
  • Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting allows for identifying and cataloguing known and novel components of glutamatergic synapses.
  • Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting identified FXYD6 and Tpd52 as novel components of glutamatergic synapses.

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