Immunoisolation and subfractionation of synaptic vesicle proteins
Immunoisolation and subfractionation of synaptic vesicle proteins
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DOI：
10.1016/j.ab.2006.12.045
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发表时间：
2007-03-15
影响因子：
2.9
通讯作者：
Volknandt, Walter
中科院分区：
文献类型：
--
作者：
Burre, Jacqueline;Zimmermann, Herbert;Volknandt, Walter
近年来，蛋白质组学技术的进步已经导致了相当多的新见解的蛋白质表达模式的特定组织，细胞和细胞器。然而，从大规模蛋白质组学方法获得的信息表明，整个细胞或组织的蛋白质组学分析通常不适合于完全解开单个细胞器成分的蛋白质组或鉴定以低拷贝数存在的蛋白质。此外，疏水蛋白的鉴定仍然是一个挑战。因此，开发适用于富集低丰度膜蛋白的技术对于全面的蛋白质组学分析至关重要。除了通过亚细胞分离富集特定的亚细胞结构外，适用于蛋白质组学研究的技术范围可以扩展到使用有机溶剂、洗涤剂和具有水溶性聚合物的基于洗涤剂的含水两相系统分离整体和外周膜蛋白。在这里，我们讨论了一些实验方案的有效性。我们表明，适当选择的物理化学条件的结果在分离的突触囊泡的高纯度，其蛋白质组可以细分成完整的膜蛋白和可溶性蛋白质的几个相分离技术。(c)2007爱思唯尔公司All rights reserved.
Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques. (c) 2007 Elsevier Inc. All rights reserved.