Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques
Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques
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DOI：
10.1002/pmic.200600357
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发表时间：
2006-12-01
期刊：
影响因子：
3.4
通讯作者：
Volknandt, Walter
中科院分区：
文献类型：
--
作者：
Burre, Jacqueline;Beckhaus, Tobias;Volknandt, Walter
突触囊泡是神经传递的关键细胞器。它们的功能由一组独特的整合和外周相关蛋白质控制。为了获得一个完整的蛋白质库存，我们免疫分离的突触囊泡从大鼠大脑的高纯度和进行基于凝胶的突触囊泡蛋白质组分析。由于整合膜蛋白的高疏水性阻碍了其通过凝胶电泳技术的分辨率，因此我们在MS之前并行应用三种不同的凝胶电泳方法用于蛋白质分离。突触囊泡蛋白进行1-D SDS-PAGE沿着纳米LC ESI-MS/MS或2-D凝胶电泳技术苄基二甲基-正十六烷基氯化铵（BAC）/SDS-PAGE，和双SDS（dSDS）-PAGE结合MALDI-TOF-MS。我们证明，所有三种方法的组合提供了一个全面的调查的突触囊泡膜室的蛋白质库存。所鉴定的突触囊泡蛋白包括转运蛋白、可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）、突触蛋白、rab和rab相互作用蛋白、另外的鸟嘌呤核苷酸三磷酸（GTP）结合蛋白、细胞骨架蛋白和调节突触囊泡胞吞和胞吞作用的蛋白。此外，我们还鉴定了一些功能未知的新蛋白质。我们的结果表明，三种不同的基于凝胶的方法与质谱法相结合的并行应用允许对突触囊泡蛋白质组进行全面的分析，这比以前预期的要复杂得多。
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.