title: Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques.
abstract: Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.
authors.affiliation: Department of Neurochemistry, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany. j.burre@cns.uni-frankfurt.de
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Burré
authors.forename: Tobias
authors.initials: T
authors.lastname: Beckhaus
authors.forename: Hermann
authors.initials: H
authors.lastname: Schägger
authors.forename: Carsten
authors.initials: C
authors.lastname: Corvey
authors.forename: Sandra
authors.initials: S
authors.lastname: Hofmann
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
authors.forename: Herbert
authors.initials: H
authors.lastname: Zimmermann
authors.forename: Walter
authors.initials: W
authors.lastname: Volknandt
mesh_terms: D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008565:Membrane Proteins / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013572:Synaptic Vesicles / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D008565:Membrane Proteins; D020543:Proteome