251. Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting
- Author
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Mads Grønborg, Henning Urlaub, Christoph Biesemann, Simon R. Bungers, Nils Brose, Elisa Luquet, Frederique Varoqueaux, Etienne Herzog, Olaf Jahn, Véronique Bernard, Ben Cooper, Sven P. Wichert, Jennifer A. Byrne, Liyi Li, Novo Nordisk A/S, Neurosciences Paris Seine (NPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute of Experimental Medicine [Göttingen] (MPI), Max-Planck-Gesellschaft, Department of Chemistry, Hong Kong Baptist University, Hong Kong Baptist University (HKBU), and The University of Sydney
- Subjects
Proteomics ,Resource ,Glutamic Acid ,Cell Separation ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Ion Channels ,Synapse ,03 medical and health sciences ,Glutamatergic ,Mice ,0302 clinical medicine ,Animals ,Molecular Biology ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,Synaptosome ,Mice, Knockout ,Neurons ,0303 health sciences ,General Immunology and Microbiology ,General Neuroscience ,Brain ,Flow Cytometry ,Cell biology ,Biochemistry ,Synapses ,Vesicular Glutamate Transport Protein 1 ,[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC] ,Glutamatergic synapse ,Cell fractionation ,Postsynaptic density ,030217 neurology & neurosurgery ,Function (biology) ,Synaptosomes - 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.
- Published
- 2014
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