Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
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用于绘制蛋白质-蛋白质相互作用网络并比较蛋白质构象状态的三功能交联剂
DOI：
10.7554/elife.12509
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发表时间：
2016-03-08
期刊：
影响因子：
7.7
通讯作者：
Lei, Xiaoguang
中科院分区：
文献类型：
--
作者：
Tan, Dan;Li, Qiang;Lei, Xiaoguang
为了提高蛋白质的化学交联联用质谱（CXMS），我们开发了一种赖氨酸靶向的可富集交联剂，其中含有用于亲和纯化的生物素标签，富集后将交联肽与生物素分离的化学裂解位点，以及可以用稳定同位素标记用于定量的间隔臂。通过定位70S核糖体表面的柔性蛋白，我们发现这种三功能交联剂可以有效地获得通过晶体学和电子显微镜难以获得的结构信息。从一个粗糙的Rrp46免疫沉淀中，它帮助鉴定了Rrp46的两个直接结合伙伴和共免疫沉淀的外泌体亚基之间的15个蛋白质-蛋白质相互作用（PPIs）。将其应用于大肠杆菌和秀丽隐杆线虫的裂解物，我们鉴定出3130和893对相互连接的赖氨酸，分别代表677和121个ppi。使用定量的CXMS工作流，我们证明它可以揭示由于蛋白质-核酸相互作用而导致赖氨酸残基反应性的变化。
To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.