Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting
Identifieur interne : 002298 ( Istex/Corpus ); précédent : 002297; suivant : 002299Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting
Auteurs : Christoph Biesemann ; Mads Gr Nborg ; Elisa Luquet ; Sven P. Wichert ; Véronique Bernard ; Simon R. Bungers ; Ben Cooper ; Frédérique Varoqueaux ; Liyi Li ; Jennifer A. Byrne ; Henning Urlaub ; Olaf Jahn ; Nils Brose ; Etienne HerzogSource :
- The EMBO Journal [ 0261-4189 ] ; 2014-01-16.
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.
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DOI: 10.1002/embj.201386120
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title><author><name sortKey="Biesemann, Christoph" sort="Biesemann, Christoph" uniqKey="Biesemann C" first="Christoph" last="Biesemann">Christoph Biesemann</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Gr Nborg, Mads" sort="Gr Nborg, Mads" uniqKey="Gr Nborg M" first="Mads" last="Gr Nborg">Mads Gr Nborg</name><affiliation><mods:affiliation>Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Current Address: Department of Beta Cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark</mods:affiliation></affiliation></author><author><name sortKey="Luquet, Elisa" sort="Luquet, Elisa" uniqKey="Luquet E" first="Elisa" last="Luquet">Elisa Luquet</name><affiliation><mods:affiliation>CNRS, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation><affiliation><mods:affiliation>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation></author><author><name sortKey="Wichert, Sven P" sort="Wichert, Sven P" uniqKey="Wichert S" first="Sven P" last="Wichert">Sven P. 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Bungers</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Cooper, Ben" sort="Cooper, Ben" uniqKey="Cooper B" first="Ben" last="Cooper">Ben Cooper</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Varoqueaux, Frederique" sort="Varoqueaux, Frederique" uniqKey="Varoqueaux F" first="Frédérique" last="Varoqueaux">Frédérique Varoqueaux</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Li, Liyi" sort="Li, Liyi" uniqKey="Li L" first="Liyi" last="Li">Liyi Li</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Byrne, Jennifer A" sort="Byrne, Jennifer A" uniqKey="Byrne J" first="Jennifer A" last="Byrne">Jennifer A. Byrne</name><affiliation><mods:affiliation>Molecular Oncology Laboratory, Children's Cancer Research Unit, Kids Research Institute, NSW, Westmead, Australia</mods:affiliation></affiliation></author><author><name sortKey="Urlaub, Henning" sort="Urlaub, Henning" uniqKey="Urlaub H" first="Henning" last="Urlaub">Henning Urlaub</name><affiliation><mods:affiliation>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Jahn, Olaf" sort="Jahn, Olaf" uniqKey="Jahn O" first="Olaf" last="Jahn">Olaf Jahn</name><affiliation><mods:affiliation>Proteomics Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Brose, Nils" sort="Brose, Nils" uniqKey="Brose N" first="Nils" last="Brose">Nils Brose</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: brose@em.mpg.de</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: etienne.herzog@u-bordeaux2.fr</mods:affiliation></affiliation></author><author><name sortKey="Herzog, Etienne" sort="Herzog, Etienne" uniqKey="Herzog E" first="Etienne" last="Herzog">Etienne Herzog</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>CNRS, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation><affiliation><mods:affiliation>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation><affiliation><mods:affiliation>INSERM U952, Université Pierre et Marie Curie, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>CNRS UMR 7224, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: brose@em.mpg.de</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: etienne.herzog@u-bordeaux2.fr</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:BB8C4FFF757259F8987D374EE777FA6BA1B24FBF</idno><date when="2014" year="2014">2014</date><idno type="doi">10.1002/embj.201386120</idno><idno type="url">https://api.istex.fr/document/BB8C4FFF757259F8987D374EE777FA6BA1B24FBF/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">002298</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002298</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title><author><name sortKey="Biesemann, Christoph" sort="Biesemann, Christoph" uniqKey="Biesemann C" first="Christoph" last="Biesemann">Christoph Biesemann</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Gr Nborg, Mads" sort="Gr Nborg, Mads" uniqKey="Gr Nborg M" first="Mads" last="Gr Nborg">Mads Gr Nborg</name><affiliation><mods:affiliation>Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Current Address: Department of Beta Cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark</mods:affiliation></affiliation></author><author><name sortKey="Luquet, Elisa" sort="Luquet, Elisa" uniqKey="Luquet E" first="Elisa" last="Luquet">Elisa Luquet</name><affiliation><mods:affiliation>CNRS, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation><affiliation><mods:affiliation>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation></author><author><name sortKey="Wichert, Sven P" sort="Wichert, Sven P" uniqKey="Wichert S" first="Sven P" last="Wichert">Sven P. Wichert</name><affiliation><mods:affiliation>Gene Expression Group, Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Current Address: Department of Psychiatry and Psychotherapy, Ludwig‐Maximilians‐University Munich, Munich, Germany</mods:affiliation></affiliation></author><author><name sortKey="Bernard, Veronique" sort="Bernard, Veronique" uniqKey="Bernard V" first="Véronique" last="Bernard">Véronique Bernard</name><affiliation><mods:affiliation>INSERM U952, Université Pierre et Marie Curie, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>CNRS UMR 7224, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</mods:affiliation></affiliation></author><author><name sortKey="Bungers, Simon R" sort="Bungers, Simon R" uniqKey="Bungers S" first="Simon R" last="Bungers">Simon R. Bungers</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Cooper, Ben" sort="Cooper, Ben" uniqKey="Cooper B" first="Ben" last="Cooper">Ben Cooper</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Varoqueaux, Frederique" sort="Varoqueaux, Frederique" uniqKey="Varoqueaux F" first="Frédérique" last="Varoqueaux">Frédérique Varoqueaux</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Li, Liyi" sort="Li, Liyi" uniqKey="Li L" first="Liyi" last="Li">Liyi Li</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Byrne, Jennifer A" sort="Byrne, Jennifer A" uniqKey="Byrne J" first="Jennifer A" last="Byrne">Jennifer A. Byrne</name><affiliation><mods:affiliation>Molecular Oncology Laboratory, Children's Cancer Research Unit, Kids Research Institute, NSW, Westmead, Australia</mods:affiliation></affiliation></author><author><name sortKey="Urlaub, Henning" sort="Urlaub, Henning" uniqKey="Urlaub H" first="Henning" last="Urlaub">Henning Urlaub</name><affiliation><mods:affiliation>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Jahn, Olaf" sort="Jahn, Olaf" uniqKey="Jahn O" first="Olaf" last="Jahn">Olaf Jahn</name><affiliation><mods:affiliation>Proteomics Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation></author><author><name sortKey="Brose, Nils" sort="Brose, Nils" uniqKey="Brose N" first="Nils" last="Brose">Nils Brose</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: brose@em.mpg.de</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: etienne.herzog@u-bordeaux2.fr</mods:affiliation></affiliation></author><author><name sortKey="Herzog, Etienne" sort="Herzog, Etienne" uniqKey="Herzog E" first="Etienne" last="Herzog">Etienne Herzog</name><affiliation><mods:affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</mods:affiliation></affiliation><affiliation><mods:affiliation>CNRS, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation><affiliation><mods:affiliation>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</mods:affiliation></affiliation><affiliation><mods:affiliation>INSERM U952, Université Pierre et Marie Curie, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>CNRS UMR 7224, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: brose@em.mpg.de</mods:affiliation></affiliation><affiliation><mods:affiliation>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: etienne.herzog@u-bordeaux2.fr</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">The EMBO Journal</title><title level="j" type="alt">THE EMBO JOURNAL</title><idno type="ISSN">0261-4189</idno><idno type="eISSN">1460-2075</idno><imprint><biblScope unit="vol">33</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="157">157</biblScope><biblScope unit="page" to="170">170</biblScope><biblScope unit="page-count">13</biblScope><date type="published" when="2014-01-16">2014-01-16</date></imprint><idno type="ISSN">0261-4189</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0261-4189</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="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.</div></front></TEI><istex><corpusName>wiley</corpusName><author><json:item><name>Christoph Biesemann</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Mads Grønborg</name><affiliations><json:string>Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany</json:string><json:string>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</json:string><json:string>Current Address: Department of Beta Cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark</json:string></affiliations></json:item><json:item><name>Elisa Luquet</name><affiliations><json:string>CNRS, IINS, UMR 5297, Bordeaux, France</json:string><json:string>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</json:string></affiliations></json:item><json:item><name>Sven P Wichert</name><affiliations><json:string>Gene Expression Group, Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string><json:string>Current Address: Department of Psychiatry and Psychotherapy, Ludwig‐Maximilians‐University Munich, Munich, Germany</json:string></affiliations></json:item><json:item><name>Véronique Bernard</name><affiliations><json:string>INSERM U952, Université Pierre et Marie Curie, Paris, France</json:string><json:string>CNRS UMR 7224, Paris, France</json:string><json:string>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</json:string></affiliations></json:item><json:item><name>Simon R Bungers</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Ben Cooper</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Frédérique Varoqueaux</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Liyi Li</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Jennifer A Byrne</name><affiliations><json:string>Molecular Oncology Laboratory, Children's Cancer Research Unit, Kids Research Institute, NSW, Westmead, Australia</json:string></affiliations></json:item><json:item><name>Henning Urlaub</name><affiliations><json:string>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</json:string><json:string>Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Olaf Jahn</name><affiliations><json:string>Proteomics Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string></affiliations></json:item><json:item><name>Nils Brose</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string><json:string>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</json:string><json:string>E-mail: brose@em.mpg.de</json:string><json:string>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</json:string><json:string>E-mail: etienne.herzog@u-bordeaux2.fr</json:string></affiliations></json:item><json:item><name>Etienne Herzog</name><affiliations><json:string>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</json:string><json:string>CNRS, IINS, UMR 5297, Bordeaux, France</json:string><json:string>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</json:string><json:string>INSERM U952, Université Pierre et Marie Curie, Paris, France</json:string><json:string>CNRS UMR 7224, Paris, France</json:string><json:string>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</json:string><json:string>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</json:string><json:string>E-mail: brose@em.mpg.de</json:string><json:string>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</json:string><json:string>E-mail: etienne.herzog@u-bordeaux2.fr</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Fluorescence Activated Synaptosome Sorting</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>proteomics</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>subcellular fractionation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>synaptosome</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>vesicular glutamate transporter</value></json:item></subject><articleId><json:string>EMBJ201386120</json:string></articleId><arkIstex>ark:/67375/WNG-NT23WBQ4-9</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>article</json:string></originalGenre><abstract>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.</abstract><qualityIndicators><refBibsNative>true</refBibsNative><abstractWordCount>151</abstractWordCount><abstractCharCount>1183</abstractCharCount><keywordCount>5</keywordCount><score>8.812</score><pdfWordCount>9746</pdfWordCount><pdfCharCount>62619</pdfCharCount><pdfVersion>1.7</pdfVersion><pdfPageCount>14</pdfPageCount><pdfPageSize>595.276 x 782.362 pts</pdfPageSize></qualityIndicators><title>Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title><genre><json:string>article</json:string></genre><host><title>The EMBO Journal</title><language><json:string>unknown</json:string></language><doi><json:string>10.1002/(ISSN)1460-2075</json:string></doi><issn><json:string>0261-4189</json:string></issn><eissn><json:string>1460-2075</json:string></eissn><publisherId><json:string>EMBJ</json:string></publisherId><volume>33</volume><issue>2</issue><pages><first>157</first><last>170</last><total>13</total></pages><genre><json:string>journal</json:string></genre><subject><json:item><value>Neuroscience</value></json:item><json:item><value>Methods & Resources</value></json:item><json:item><value>Resource</value></json:item></subject></host><ark><json:string>ark:/67375/WNG-NT23WBQ4-9</json:string></ark><publicationDate>2014</publicationDate><copyrightDate>2014</copyrightDate><doi><json:string>10.1002/embj.201386120</json:string></doi><id>BB8C4FFF757259F8987D374EE777FA6BA1B24FBF</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/document/BB8C4FFF757259F8987D374EE777FA6BA1B24FBF/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/document/BB8C4FFF757259F8987D374EE777FA6BA1B24FBF/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/BB8C4FFF757259F8987D374EE777FA6BA1B24FBF/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>Wiley Publishing Ltd</publisher><availability><licence>© 2013 The Authors</licence></availability><date type="published" when="2014-01-16"></date></publicationStmt><notesStmt><note type="content-type" subtype="article" source="article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</note><note type="publication-type" subtype="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title><author xml:id="author-0000"><persName><forename type="first">Christoph</forename><surname>Biesemann</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0001"><persName><forename type="first">Mads</forename><surname>Grønborg</surname></persName><affiliation><orgName>Department of Neurobiology</orgName><orgName>Max Planck Institute of Biophysical Chemistry</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation><affiliation><orgName>Bioanalytical Mass Spectrometry</orgName><orgName>Max Planck Institute for Biophysical Chemistry</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation><affiliation>Current Address: <orgName>Department of Beta Cell Regeneration</orgName><orgName>Hagedorn Research Institute</orgName><address><settlement type="city">Gentofte</settlement><country key="DK">Denmark</country></address></affiliation></author><author xml:id="author-0002"><persName><forename type="first">Elisa</forename><surname>Luquet</surname></persName><affiliation><orgName>CNRS</orgName><orgName>IINS</orgName><orgName>UMR 5297</orgName><address><settlement type="city">Bordeaux</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>University of Bordeaux</orgName><orgName>IINS</orgName><orgName>UMR 5297</orgName><address><settlement type="city">Bordeaux</settlement><country key="FR">France</country></address></affiliation></author><author xml:id="author-0003"><persName><forename type="first">Sven P</forename><surname>Wichert</surname></persName><affiliation><orgName>Gene Expression Group</orgName><orgName>Department of Neurogenetics</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation><affiliation>Current Address: <orgName>Department of Psychiatry and Psychotherapy</orgName><orgName>Ludwig‐Maximilians‐University Munich</orgName><address><settlement type="city">Munich</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0004"><persName><forename type="first">Véronique</forename><surname>Bernard</surname></persName><affiliation><orgName>INSERM U952</orgName><orgName>Université Pierre et Marie Curie</orgName><address><settlement type="city">Paris</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>CNRS UMR 7224</orgName><address><settlement type="city">Paris</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>Université Pierre et Marie Curie (UPMC) Paris 06</orgName><orgName>PMSNC</orgName><address><settlement type="city">Paris</settlement><country key="FR">France</country></address></affiliation></author><author xml:id="author-0005"><persName><forename type="first">Simon R</forename><surname>Bungers</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0006"><persName><forename type="first">Ben</forename><surname>Cooper</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0007"><persName><forename type="first">Frédérique</forename><surname>Varoqueaux</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0008"><persName><forename type="first">Liyi</forename><surname>Li</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0009"><persName><forename type="first">Jennifer A</forename><surname>Byrne</surname></persName><affiliation><orgName>Molecular Oncology Laboratory</orgName><orgName>Children's Cancer Research Unit</orgName><orgName>Kids Research Institute</orgName><address><settlement type="city">Westmead</settlement><region>NSW</region><country key="AU">Australia</country></address></affiliation></author><author xml:id="author-0010"><persName><forename type="first">Henning</forename><surname>Urlaub</surname></persName><affiliation><orgName>Bioanalytical Mass Spectrometry</orgName><orgName>Max Planck Institute for Biophysical Chemistry</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation><affiliation><orgName>Bioanalytics</orgName><orgName>Department of Clinical Chemistry</orgName><orgName>University Medical Center Göttingen</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0011"><persName><forename type="first">Olaf</forename><surname>Jahn</surname></persName><affiliation><orgName>Proteomics Group</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation></author><author xml:id="author-0012" role="corresp"><persName><forename type="first">Nils</forename><surname>Brose</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation><affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail: brose@em.mpg.de Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail: etienne.herzog@u-bordeaux2.fr</affiliation></author><author xml:id="author-0013" role="corresp"><persName><forename type="first">Etienne</forename><surname>Herzog</surname></persName><affiliation><orgName>Department of Molecular Neurobiology</orgName><orgName>Max Planck Institute of Experimental Medicine</orgName><address><settlement type="city">Göttingen</settlement><country key="DE">Germany</country></address></affiliation><affiliation><orgName>CNRS</orgName><orgName>IINS</orgName><orgName>UMR 5297</orgName><address><settlement type="city">Bordeaux</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>University of Bordeaux</orgName><orgName>IINS</orgName><orgName>UMR 5297</orgName><address><settlement type="city">Bordeaux</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>INSERM U952</orgName><orgName>Université Pierre et Marie Curie</orgName><address><settlement type="city">Paris</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>CNRS UMR 7224</orgName><address><settlement type="city">Paris</settlement><country key="FR">France</country></address></affiliation><affiliation><orgName>Université Pierre et Marie Curie (UPMC) Paris 06</orgName><orgName>PMSNC</orgName><address><settlement type="city">Paris</settlement><country key="FR">France</country></address></affiliation><affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail: brose@em.mpg.de Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail: etienne.herzog@u-bordeaux2.fr</affiliation></author><idno type="istex">BB8C4FFF757259F8987D374EE777FA6BA1B24FBF</idno><idno type="ark">ark:/67375/WNG-NT23WBQ4-9</idno><idno type="DOI">10.1002/embj.201386120</idno><idno type="unit">EMBJ201386120</idno><idno type="toTypesetVersion">file:EMBJ.EMBJ201386120.pdf</idno></analytic><monogr><title level="j" type="main">The EMBO Journal</title><title level="j" type="alt">THE EMBO JOURNAL</title><idno type="pISSN">0261-4189</idno><idno type="eISSN">1460-2075</idno><idno type="book-DOI">10.1002/(ISSN)1460-2075</idno><idno type="book-part-DOI">10.1002/embj.v33.2</idno><idno type="product">EMBJ</idno><imprint><biblScope unit="vol">33</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="157">157</biblScope><biblScope unit="page" to="170">170</biblScope><biblScope unit="page-count">13</biblScope><date type="published" when="2014-01-16"></date></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><abstract style="main" xml:id="embj201386120-abs-0001"><head>Abstract</head><p>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 (<hi rend="fc">FASS</hi>) 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 <hi rend="fc">FASS</hi> isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. <hi rend="fc">FASS</hi> synaptosomes contain <hi rend="italic">bona fide</hi> glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in <hi rend="fc">FASS</hi> samples, of which <hi rend="fc">FXYD</hi>6 and Tpd52 were validated as new synaptic proteins. <hi rend="fc">FASS</hi> purification thus enables high‐resolution biochemical analyses of specific synapse subpopulations in health and disease.</p></abstract><abstract xml:lang="en" style="synopsis" xml:id="embj201386120-abs-0002"><head>Synopsis</head><p><figure type="box"><media mimeType="image" url="urn:x-wiley:02614189:media:embj201386120:embj201386120-abs-0001"></media></figure></p><p>This study describes the establishment, validation, and application of a new fluorescence‐activated sorting method ‐ Fluorescence Activated Synaptosome Sorting or <hi rend="fc">FASS</hi> ‐ that allows for purification of glutamatergic synaptosomes from knock‐in mutant mice expressing the fluorescent synaptic marker protein <hi rend="fc">VGLUT</hi>1‐Venus.
<list xml:id="embj201386120-list-0001" style="bulleted"><item>Fluorescence Activated Synaptosome Sorting from samples of <hi rend="fc">VGLUT</hi>1‐Venus knock‐in mice enriches glutamatergic synaptosomes to near homogeneity.</item><item>Glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting contain <hi rend="italic">bona fide</hi> glutamatergic synapse proteins but lack other synapse types and glia cell contaminants.</item><item>Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting allows for identifying and cataloguing known and novel components of glutamatergic synapses. </item><item>Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting identified <hi rend="fc">FXYD</hi>6 and Tpd52 as novel components of glutamatergic synapses.</item></list></p></abstract><abstract style="graphical" xml:id="embj201386120-abs-0003"><p>A novel Fluorescence Activated Synaptosome Sorting (FASS) approach allows for high‐resolution biochemical analyses of specific synapse subpopulations and identification of 163 proteins that are specifically enriched in FASS‐sorted synaptosomes.<figure type="box"><media mimeType="image" url="urn:x-wiley:02614189:embj201386120:embj201386120-toc-0001"></media></figure></p></abstract><textClass><keywords><term xml:id="embj201386120-kwd-0001">Fluorescence Activated Synaptosome Sorting</term><term xml:id="embj201386120-kwd-0002">proteomics</term><term xml:id="embj201386120-kwd-0003">subcellular fractionation</term><term xml:id="embj201386120-kwd-0004">synaptosome</term><term xml:id="embj201386120-kwd-0005">vesicular glutamate transporter</term></keywords><classCode scheme="http://psi.embo.org/14602075/EMBO27">Neuroscience</classCode><classCode scheme="http://psi.embo.org/14602075/EMBO22">Methods & Resources</classCode><keywords rend="articleCategory"><term>Resource</term></keywords><keywords rend="tocHeading1"><term>Resource</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component type="serialArticle" version="2.0" xml:id="embj201386120" xml:lang="en"><header><publicationMeta level="product"><doi origin="wiley" registered="yes">10.1002/(ISSN)1460-2075</doi><issn type="print">0261-4189</issn><issn type="electronic">1460-2075</issn><idGroup><id type="product" value="EMBJ"></id></idGroup><titleGroup><title sort="THE EMBO JOURNAL" type="main">The EMBO Journal</title><title type="short">EMBO J</title></titleGroup></publicationMeta><publicationMeta level="part" position="20"><doi origin="wiley">10.1002/embj.v33.2</doi><copyright ownership="thirdParty">Copyright © 2014 EMBO</copyright><numberingGroup><numbering type="journalVolume" number="33">33</numbering><numbering type="journalIssue">2</numbering></numberingGroup><coverDate startDate="2014-01-16">16 January 2014</coverDate></publicationMeta><publicationMeta level="unit" position="80" status="forIssue" type="article"><doi>10.1002/embj.201386120</doi><idGroup><id type="unit" value="EMBJ201386120"></id></idGroup><countGroup><count number="13" type="pageTotal"></count></countGroup><titleGroup><title type="articleCategory">Resource</title><title type="tocHeading1">Resource</title></titleGroup><copyright ownership="thirdParty">© 2013 The Authors</copyright><eventGroup><event date="2013-06-28" type="manuscriptReceived"></event><event date="2013-11-10" type="manuscriptRevised"></event><event date="2013-11-18" type="manuscriptAccepted"></event><event agent="SPS" date="2013-11-28" type="xmlCreated"></event><event agent="SPS" date="2014-06-24" type="xmlCorrected"></event><event type="publishedOnlineEarlyUnpaginated" date="2014-01-10"></event><event type="publishedOnlineFinalForm" date="2014-01-14"></event><event type="firstOnline" date="2014-01-10"></event><event type="xmlConverted" agent="Converter:WML3G_To_WML3G version:4.6.4 mode:FullText" date="2015-10-08"></event></eventGroup><numberingGroup><numbering type="pageFirst">157</numbering><numbering type="pageLast">170</numbering></numberingGroup><correspondenceTo><lineatedText><line>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail: <email>brose@em.mpg.de</email></line></lineatedText><lineatedText><line>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail: <email>etienne.herzog@u-bordeaux2.fr</email></line></lineatedText></correspondenceTo><subjectInfo><subject href="http://psi.embo.org/14602075/EMBO27">Neuroscience</subject><subject href="http://psi.embo.org/14602075/EMBO22">Methods & Resources</subject></subjectInfo><selfCitationGroup><citation type="self" xml:id="embj201283326-cit-1001"><journalTitle>EMBO Journal</journalTitle> (<pubYear year="2014">2014</pubYear>) <vol>33</vol>, <pageFirst>157</pageFirst>–<pageLast>170</pageLast></citation></selfCitationGroup><linkGroup><link type="toTypesetVersion" href="file:EMBJ.EMBJ201386120.pdf"></link></linkGroup></publicationMeta><contentMeta><titleGroup><title type="main">Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title><title type="shortAuthors">Christoph Biesemann et al</title></titleGroup><creators><creator affiliationRef="#embj201386120-aff-0001" creatorRole="author" 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<country>France</country></address></affiliation><affiliation countryCode="FR" type="organization" xml:id="embj201386120-aff-0008"><orgName>Université Pierre et Marie Curie (UPMC) Paris 06</orgName> <orgName>PMSNC</orgName> <address><city>Paris</city> <country>France</country></address></affiliation><affiliation countryCode="AU" type="organization" xml:id="embj201386120-aff-0009"><orgDiv>Molecular Oncology Laboratory</orgDiv> <orgDiv>Children's Cancer Research Unit</orgDiv> <orgName>Kids Research Institute</orgName> <address><city>Westmead</city> <countryPart>NSW</countryPart> <country>Australia</country></address></affiliation><affiliation countryCode="DE" type="organization" xml:id="embj201386120-aff-0010"><orgDiv>Bioanalytical Mass Spectrometry</orgDiv> <orgName>Max Planck Institute for Biophysical Chemistry</orgName> <address><city>Göttingen</city> <country>Germany</country></address></affiliation><affiliation countryCode="DE" type="organization" xml:id="embj201386120-aff-0011"><orgDiv>Bioanalytics</orgDiv> <orgDiv>Department of Clinical Chemistry</orgDiv> <orgName>University Medical Center Göttingen</orgName> <address><city>Göttingen</city> <country>Germany</country></address></affiliation><affiliation countryCode="DE" type="organization" xml:id="embj201386120-aff-0012"><orgDiv>Proteomics Group</orgDiv> <orgName>Max Planck Institute of Experimental Medicine</orgName> <address><city>Göttingen</city> <country>Germany</country></address></affiliation><affiliation countryCode="DK" type="organization" xml:id="embj201386120-curr-0001"><orgDiv>Department of Beta Cell Regeneration</orgDiv> <orgName>Hagedorn Research Institute</orgName> <address><city>Gentofte</city> <country>Denmark</country></address></affiliation><affiliation countryCode="DE" type="organization" xml:id="embj201386120-curr-0002"><orgDiv>Department of Psychiatry and Psychotherapy</orgDiv> <orgName>Ludwig‐Maximilians‐University Munich</orgName> <address><city>Munich</city> <country>Germany</country></address></affiliation></affiliationGroup><keywordGroup type="author"><keyword xml:id="embj201386120-kwd-0001">Fluorescence Activated Synaptosome Sorting</keyword><keyword xml:id="embj201386120-kwd-0002">proteomics</keyword><keyword xml:id="embj201386120-kwd-0003">subcellular fractionation</keyword><keyword xml:id="embj201386120-kwd-0004">synaptosome</keyword><keyword xml:id="embj201386120-kwd-0005">vesicular glutamate transporter</keyword></keywordGroup><fundingInfo><fundingAgency>European Union</fundingAgency></fundingInfo><fundingInfo><fundingAgency>Max Planck Society</fundingAgency></fundingInfo><fundingInfo><fundingAgency>German Research Foundation</fundingAgency><fundingNumber>GRK521</fundingNumber></fundingInfo><fundingInfo><fundingAgency>Agence Nationale de la Recherche</fundingAgency><fundingNumber>ANR‐12‐JSV4‐0005‐01 VGLUT‐IQ</fundingNumber><fundingNumber>ANR‐10‐LABX‐43 BRAIN</fundingNumber></fundingInfo><supportingInformation><supportingInfoItem xml:id="embj201386120-sup-0001"><mediaResource alt="supporting" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0001-FigS1"></mediaResource><caption>Supplementary Figure S1</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0002"><mediaResource alt="supporting" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0002-FigS2"></mediaResource><caption>Supplementary Figure S2</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0003"><mediaResource alt="supporting" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0003-FigS3"></mediaResource><caption>Supplementary Figure S3</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0004"><mediaResource alt="supporting" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0004-FigS4"></mediaResource><caption>Supplementary Figure S4</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0005"><mediaResource alt="supporting" mimeType="application/msexcel" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0005-TableS1"></mediaResource><caption>Supplementary Table S1</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0006"><mediaResource alt="supporting" mimeType="application/msexcel" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0006-TableS2"></mediaResource><caption>Supplementary Table S2</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0007"><mediaResource alt="supporting" mimeType="application/msexcel" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0007-TableS3"></mediaResource><caption>Supplementary Table S3</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0008"><mediaResource alt="supporting" mimeType="application/msexcel" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0008-TableS4"></mediaResource><caption>Supplementary Table S4</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0009"><mediaResource alt="supporting" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201386120:embj201386120-sup-0009-DataS1"></mediaResource><caption>Supplementary Data S1</caption></supportingInfoItem><supportingInfoItem xml:id="embj201386120-sup-0010"><mediaResource alt="supporting" mimeType="application/pdf" href="urn-x:wiley:02614189:media:embj201386120:embj201386120"></mediaResource><caption>Review Process File</caption></supportingInfoItem></supportingInformation><abstractGroup><abstract type="main" xml:id="embj201386120-abs-0001"><title type="main">Abstract</title><p>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 (<fc>FASS</fc>) 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 <fc>FASS</fc> isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. <fc>FASS</fc> synaptosomes contain <i>bona fide</i> glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in <fc>FASS</fc> samples, of which <fc>FXYD</fc>6 and Tpd52 were validated as new synaptic proteins. <fc>FASS</fc> purification thus enables high‐resolution biochemical analyses of specific synapse subpopulations in health and disease.</p></abstract><abstract type="synopsis" xml:id="embj201386120-abs-0002" xml:lang="en"><title type="main">Synopsis</title><p><blockFixed type="graphic" xml:id="embj201386120-blkfxd-0001"><mediaResourceGroup><mediaResource alt="image" href="urn:x-wiley:02614189:media:embj201386120:embj201386120-abs-0001"></mediaResource></mediaResourceGroup></blockFixed></p><p>This study describes the establishment, validation, and application of a new fluorescence‐activated sorting method ‐ Fluorescence Activated Synaptosome Sorting or <fc>FASS</fc> ‐ that allows for purification of glutamatergic synaptosomes from knock‐in mutant mice expressing the fluorescent synaptic marker protein <fc>VGLUT</fc>1‐Venus.
<list formatted="paragraph" style="bulleted" xml:id="embj201386120-list-0001"><listItem>Fluorescence Activated Synaptosome Sorting from samples of <fc>VGLUT</fc>1‐Venus knock‐in mice enriches glutamatergic synaptosomes to near homogeneity.</listItem><listItem>Glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting contain <i>bona fide</i> glutamatergic synapse proteins but lack other synapse types and glia cell contaminants.</listItem><listItem>Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting allows for identifying and cataloguing known and novel components of glutamatergic synapses. </listItem><listItem>Proteomic analysis of glutamatergic synaptosomes purified by Fluorescence Activated Synaptosome Sorting identified <fc>FXYD</fc>6 and Tpd52 as novel components of glutamatergic synapses.</listItem></list></p></abstract><abstract type="graphical" xml:id="embj201386120-abs-0003"><p>A novel Fluorescence Activated Synaptosome Sorting (FASS) approach allows for high‐resolution biochemical analyses of specific synapse subpopulations and identification of 163 proteins that are specifically enriched in FASS‐sorted synaptosomes.<blockFixed type="graphic" xml:id="embj201386120-blkfxd-0002"><mediaResourceGroup><mediaResource alt="image" href="urn:x-wiley:02614189:embj201386120:embj201386120-toc-0001"></mediaResource></mediaResourceGroup></blockFixed></p></abstract></abstractGroup></contentMeta></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting</title></titleInfo><name type="personal"><namePart type="given">Christoph</namePart><namePart type="family">Biesemann</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Mads</namePart><namePart type="family">Grønborg</namePart><affiliation>Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany</affiliation><affiliation>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</affiliation><affiliation>Current Address: Department of Beta Cell Regeneration, Hagedorn Research Institute, Gentofte, Denmark</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Elisa</namePart><namePart type="family">Luquet</namePart><affiliation>CNRS, IINS, UMR 5297, Bordeaux, France</affiliation><affiliation>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Sven P</namePart><namePart type="family">Wichert</namePart><affiliation>Gene Expression Group, Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><affiliation>Current Address: Department of Psychiatry and Psychotherapy, Ludwig‐Maximilians‐University Munich, Munich, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Véronique</namePart><namePart type="family">Bernard</namePart><affiliation>INSERM U952, Université Pierre et Marie Curie, Paris, France</affiliation><affiliation>CNRS UMR 7224, Paris, France</affiliation><affiliation>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Simon R</namePart><namePart type="family">Bungers</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Ben</namePart><namePart type="family">Cooper</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Frédérique</namePart><namePart type="family">Varoqueaux</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Liyi</namePart><namePart type="family">Li</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Jennifer A</namePart><namePart type="family">Byrne</namePart><affiliation>Molecular Oncology Laboratory, Children's Cancer Research Unit, Kids Research Institute, NSW, Westmead, Australia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Henning</namePart><namePart type="family">Urlaub</namePart><affiliation>Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany</affiliation><affiliation>Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Olaf</namePart><namePart type="family">Jahn</namePart><affiliation>Proteomics Group, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Nils</namePart><namePart type="family">Brose</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</affiliation><affiliation>E-mail: brose@em.mpg.de</affiliation><affiliation>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</affiliation><affiliation>E-mail: etienne.herzog@u-bordeaux2.fr</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Etienne</namePart><namePart type="family">Herzog</namePart><affiliation>Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany</affiliation><affiliation>CNRS, IINS, UMR 5297, Bordeaux, France</affiliation><affiliation>University of Bordeaux, IINS, UMR 5297, Bordeaux, France</affiliation><affiliation>INSERM U952, Université Pierre et Marie Curie, Paris, France</affiliation><affiliation>CNRS UMR 7224, Paris, France</affiliation><affiliation>Université Pierre et Marie Curie (UPMC) Paris 06, PMSNC, Paris, France</affiliation><affiliation>Corresponding author. Tel: +49 551 3899 725; Fax: +49 551 3899 715; E‐mail:</affiliation><affiliation>E-mail: brose@em.mpg.de</affiliation><affiliation>Corresponding author. Tel: +33 5 57 57 57 48; Fax: +33 5 57 57 40 82; E‐mail:</affiliation><affiliation>E-mail: etienne.herzog@u-bordeaux2.fr</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-6N5SZHKN-D">article</genre><originInfo><publisher>Blackwell Publishing Ltd</publisher><dateIssued encoding="w3cdtf">2014-01-16</dateIssued><dateCreated encoding="w3cdtf">2013-11-28</dateCreated><dateCaptured encoding="w3cdtf">2013-06-28</dateCaptured><dateValid encoding="w3cdtf">2013-11-18</dateValid><edition>EMBO Journal (2014) 33, 157–170</edition><copyrightDate encoding="w3cdtf">2014</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><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.</abstract><abstract type="synopsis" lang="en">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.</abstract><abstract type="graphical">A novel Fluorescence Activated Synaptosome Sorting (FASS) approach allows for high‐resolution biochemical analyses of specific synapse subpopulations and identification of 163 proteins that are specifically enriched in FASS‐sorted synaptosomes.</abstract><note type="additional physical form">Supplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Data S1Review Process File</note><note type="funding">European Union</note><note type="funding">Max Planck Society</note><note type="funding">German Research Foundation - No. GRK521; </note><note type="funding">Agence Nationale de la Recherche - No. ANR‐12‐JSV4‐0005‐01 VGLUT‐IQ; No. ANR‐10‐LABX‐43 BRAIN; </note><subject><genre>keywords</genre><topic>Fluorescence Activated Synaptosome Sorting</topic><topic>proteomics</topic><topic>subcellular fractionation</topic><topic>synaptosome</topic><topic>vesicular glutamate transporter</topic></subject><relatedItem type="host"><titleInfo><title>The EMBO Journal</title></titleInfo><titleInfo type="abbreviated"><title>EMBO J</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><subject><genre>index-terms</genre><topic authorityURI="http://psi.embo.org/14602075/EMBO27">Neuroscience</topic><topic authorityURI="http://psi.embo.org/14602075/EMBO22">Methods & Resources</topic></subject><subject><genre>article-category</genre><topic>Resource</topic></subject><identifier type="ISSN">0261-4189</identifier><identifier type="eISSN">1460-2075</identifier><identifier type="DOI">10.1002/(ISSN)1460-2075</identifier><identifier type="PublisherID">EMBJ</identifier><part><date>2014</date><detail type="volume"><caption>vol.</caption><number>33</number></detail><detail type="issue"><caption>no.</caption><number>2</number></detail><extent unit="pages"><start>157</start><end>170</end><total>13</total></extent></part></relatedItem><identifier type="istex">BB8C4FFF757259F8987D374EE777FA6BA1B24FBF</identifier><identifier type="ark">ark:/67375/WNG-NT23WBQ4-9</identifier><identifier type="DOI">10.1002/embj.201386120</identifier><identifier type="ArticleID">EMBJ201386120</identifier><accessCondition type="use and reproduction" contentType="copyright">Copyright © 2014 EMBO© 2013 The Authors</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-L0C46X92-X">wiley</recordContentSource></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/document/BB8C4FFF757259F8987D374EE777FA6BA1B24FBF/metadata/json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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