Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.
Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.
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DOI：
10.1091/mbc.e14-07-1198
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发表时间：
2014-10-15
影响因子：
3.3
通讯作者：
Robinson MS
中科院分区：
文献类型：
--
作者：
Borner GH;Hein MY;Hirst J;Edgar JR;Mann M;Robinson MS
分离谱分析是膜囊泡和蛋白质颗粒快速蛋白质组学表征的通用方法。虽然最初是为了分析网格蛋白包被的囊泡而开发的，但它也为生成高分辨率蛋白质相互作用图提供了一个多功能工具，允许对蛋白质复合物进行详细分析。我们开发了“分离分析”，这是一种快速分析膜囊泡和蛋白质颗粒的蛋白质组学方法。该方法将定量蛋白质组学与亚细胞分离相结合，生成特征蛋白丰度分布概况。通过聚类分析揭示了蛋白质的功能相关组。为了验证该方法，我们首先分析了HeLa细胞中的>3500蛋白，并以>90%的准确率鉴定了已知的网格蛋白包被的囊泡蛋白。然后，我们从果蝇S2细胞中分析了bbbb2400个蛋白，并报道了第一个全面的昆虫网格蛋白包被囊泡蛋白质组。重要的是，聚类分析扩展到所有的蛋白质，从而确定了各种已知的和新的细胞质和膜相关蛋白质复合物。我们表明，它也允许配合物的详细组成表征，包括亚配合物和亚基化学计量的描绘。我们的预测呈现在一个交互式数据库中。分离谱分析是一种用于定义网格蛋白包被的囊泡蛋白质组的通用方法，可用于分析其他类型的囊泡和颗粒。此外，它还为快速生成大规模蛋白质相互作用图提供了一个多功能工具。
Fractionation profiling is a universal method for the rapid proteomic characterization of membrane vesicles and protein particles. Although initially developed for the analysis of clathrin-coated vesicles, it also provides a versatile tool for generating high-resolution protein-interaction maps, allowing detailed analysis of protein complexes. We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.