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

EMBO J. 2014 Jan 13;33(2):157-70. doi: 10.1002/embj.201386120. Epub 2014 Jan 10.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / cytology*
  • Brain / metabolism
  • Cell Separation / methods
  • Flow Cytometry / methods*
  • Glutamic Acid / metabolism*
  • Ion Channels / metabolism
  • Mice
  • Mice, Knockout
  • Neurons / cytology*
  • Neurons / metabolism
  • Proteomics
  • Synapses / metabolism
  • Synaptosomes / physiology*
  • Vesicular Glutamate Transport Protein 1 / genetics
  • Vesicular Glutamate Transport Protein 1 / metabolism

Substances

  • FXYD6 protein, mouse
  • Ion Channels
  • Slc17a7 protein, mouse
  • Vesicular Glutamate Transport Protein 1
  • Glutamic Acid