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Analysis of the synaptic vesicle proteome using three gel‐based protein separation techniques
Author(s) -
Burré Jacqueline,
Beckhaus Tobias,
Schägger Hermann,
Corvey Carsten,
Hofmann Sandra,
Karas Michael,
Zimmermann Herbert,
Volknandt Walter
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200600357
Subject(s) - synaptic vesicle , proteome , rab , vesicle , kiss and run fusion , vesicle fusion , synaptic vesicle recycling , membrane protein , vesicular transport protein , biology , snap25 , neurotransmission , biochemistry , synapse , chemistry , microbiology and biotechnology , gtpase , receptor , membrane , neuroscience
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.