Trapping mammalian protein complexes in viral particles.
Trapping mammalian protein complexes in viral particles.
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DOI：
10.1038/ncomms11416
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发表时间：
2016-04-28
影响因子：
16.6
通讯作者：
Tavernier J
中科院分区：
文献类型：
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作者：
Eyckerman S;Titeca K;Van Quickelberghe E;Cloots E;Verhee A;Samyn N;De Ceuninck L;Timmerman E;De Sutter D;Lievens S;Van Calenbergh S;Gevaert K;Tavernier J
细胞裂解是经典的基于质谱的蛋白质复合物分析策略中不可避免的步骤。目前使用互补裂解条件、原位交联策略和近端标记技术来减少对蛋白质复合物的裂解作用。我们开发了Virotrap，一种病毒颗粒分选方法，无需细胞均质化，并在纯化过程中保留蛋白质复合物。通过将诱饵蛋白融合到HIV-1 GAG蛋白，我们发现相互作用伙伴被困在从哺乳动物细胞出芽的病毒样颗粒（VLP）中。使用有效的VLP富集方案，Virotrap允许检测已知的二元相互作用和基于MS的新蛋白质伴侣的鉴定。此外，我们显示的刺激依赖的相互作用的识别和演示捕获的蛋白质合作伙伴的小分子。Virotrap构成了一个优雅的补充方法的武器库的方法来研究蛋白质复合物。 蛋白质组的大部分作为大分子复合物的一部分执行其细胞功能。在这里，作者描述了Virotrap，一种新的无裂解方法，用于分离和鉴定生物相关的蛋白质-蛋白质和小分子-蛋白质相互作用。
Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. A large portion of the proteome carries out its cellular function as part of macromolecular complexes. Here the authors describe Virotrap, a novel lysis-free approach for the isolation and identification of biologically relevant protein-protein and small molecule-protein interactions.