Synaptic Vesicle Proteome of Rat Brain Bull. Korean Chem. Soc. 2007, Vol. 28, No. 9 1499 A Comprehensive Identification of Synaptic Vesicle Proteins in Rat Brains by cRPLC/MS-MS and 2DE/MALDI-TOF-MS Won-Kyu Lee, 1,2,a Hye-Jung Kim, 1,3,a Hye-Ki Min, 4 Un-Beom Kang, 1 Cheolju Lee, 1 Sang-Won Lee, 4 Ick Young Kim, 2 Seung-Taek Lee, 3 Oh-Seung Kwon, 1 and Yeon Gyu Yu 5,* 1 Division of Life Sciences, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Korea 2 Laboratory of Cellular and Molecular Biochemistry, School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea 3 Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea 4 Department of Chemistry, Korea University, Seoul 136-701, Korea 5 Department of Chemistry, Kookmin University, Seoul 136-702, Korea. * E-mail: ygyu@kookmin.ac.kr Received May 10, 2007 Proteomic analyses of synaptic vesicle fraction from rat brain have been performed for the better understanding of vesicle regulation and signal transmission. Two different approaches were applied to identify proteins in synaptic vesicle fraction. First, the isolated synaptic vesicle proteins were treated with trypsin, and the resulting peptides were analyzed using a high-pressure capillary reversed phase liquid chromatography/tandem mass spectrometry (cRPLC/MS/MS). Alternatively, proteins were separated by two-dimensional gel electrophoresis (2DE) and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS). Total 18 and 52 proteins were identified from cRPLC/MS-MS and 2DE-MALDI-TOF-MS analysis, respectively. Among them only 2 proteins were identified by both methods. Of the proteins identified, 70% were soluble proteins and 30% were membrane proteins. They were categorized by their functions in vesicle trafficking and biogenesis, energy metabolism, signal transduction, transport and unknown functions. Among them, 27 proteins were not previously reported as synaptic proteins. The cellular functions of unknown proteins were estimated from the analysis of domain structure, expression profile and predicted interaction partners. Key Words : Synaptic membrane proteome, Micro liquid chromatography electrospray ionization tandem mass spectrometry, Two-dimensional gel electrophoresis, Matrix-assisted laser desorption mass spectrometry, Rat brains Introduction Neuronal transmission in the central nervous system occurs at synaptic junctions. Incoming electronic signals from pre-synaptic neurons induce the release of neuro- transmitters in synaptic vesicles to synaptic junction, and then the released neurotransmitters bind to corresponding receptors to activate the postsynaptic neurons. Synaptic vesicles are the key organelles involved in synaptic func- tions such as uptake, storage and stimulus-dependent release of neurotransmitters. 1,2 The trafficking cycle of synaptic vesicles consists of the transport of neurotransmitters into synaptic vesicles, clustering and docking of the vesicle in front of the active zone, priming and fusion of the vesicle with the plasma membrane and recycling by endocytosis. Proteins involved in the biogenesis and transport process of synaptic vesicles have been identified from genetic as well as biochemical analyses. The synaptosome-associated protein receptor (SNARE) complex, consisting syntaxin, 3 synaptobrevin (VAMP), SNAP25, 4 and synaptotagamins, 5 is necessary for the docking and fusion of synaptic vesicles with the plasma membrane. Cytoskeleton proteins, such as myosin-V, rab 6 and KIF3A, are involved in the transport of vesicle through the cytoplasm. 6 Septins are involved in the targeting of vesicles to plasma membrane. 7,8 Proteins impli- cated in the attachment of vesicle to acceptor membranes are Rab, 9,10 rabkinesin, 11 rabadaptin, 12,13 rabphilin, 14-16 Rim, 17 EEA1 18,19 and Uso1p/p115. 20,21 The vATPase complex, which provides energy for sequestering neurotransmitters in synaptic vesicles, is also highly expressed in the synaptic vesicle. 22 In addition, transporters, receptors, and receptor- associated proteins had been identified in synapse. 23 Recently, global analyses of proteins from whole organism, specific tissues or organelles have been performed due to the recent advance in high-throughput mass spectrometric techniques. 24-26 Proteomes of human and mouse brain tissues have been analyzed using 2DE and MALDI-TOF-MS, and 200-450 proteins were identified. 27-32 Among them, only a few proteins involved in the trafficking of synaptic vesicles or signal transduction were identified. 33 Analyses of proteins in subcellular fraction from neuronal tissue, such as synapto- some or synaptic vesicles, were also carried out to identify proteins directly associated with synaptic vesicles. 34-36 Also, synaptosomal proteins from squid optic lobe have been separated using 2DE, and highly expressed proteins were a These authors equally contributed to this work. Abbreviations: 2DE, two-dimensional gel electrophoresis; ESI, elec- trospray ionization; MALDI, matrix-assisted laser desorption ioniza- tion; TOF, time-of-flight; cRPLC, capillary reversed phase liquid chromatography; QIT, quadrupole ion trap; IEF, isoelectric focusing