Capture and mass spectrometry analysis of effector-substrate complexes using genetically incorporated photo-crosslinkers in host cells.
Capture and mass spectrometry analysis of effector-substrate complexes using genetically incorporated photo-crosslinkers in host cells.
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使用宿主细胞中基因整合的光交联剂捕获和质谱分析效应子-底物复合物
DOI：
10.1016/j.xpro.2022.101882
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发表时间：
2022-12-16
期刊：
影响因子：
--
通讯作者：
Ren, Haiyan
中科院分区：
文献类型：
--
作者：
Li, Pan;Li, Jingxiang;Ren, Haiyan
效应器和宿主目标之间的相互作用通常是微弱的或短暂的，使得它们很难识别。我们描述了一种使用遗传密码扩展技术在活细胞中共价捕获效应器底物的协议。效应器-底物复合体被交联剂捕获，然后用串联层析进行纯化。我们详细介绍了质谱分析和衬底验证的步骤。虽然这里的步骤是针对HEK293T细胞中的肠源性大肠杆菌底物的，但该协议有更广泛的应用。有关本议定书使用和执行的完整细节，请参考Li等人的意见。(2021年)。使用特定部位的、结合的光交联剂的共价捕获识别活的宿主细胞中病原体的弱或瞬时相互作用步骤用于质谱分析和底物验证适用于识别除效应器以外的蛋白质的相互作用伙伴出版者注：进行任何实验方案都需要遵守当地实验室安全和伦理的机构指南。效应器和宿主目标之间的相互作用通常是微弱的或短暂的，使得它们很难识别。我们描述了一种使用遗传密码扩展技术在活细胞中共价捕获效应器底物的协议。效应器-底物复合体被交联剂捕获，然后用串联层析进行纯化。我们详细介绍了质谱分析和衬底验证的步骤。虽然这里的步骤是针对HEK293T细胞中的肠源性大肠杆菌底物的，但该协议有更广泛的应用。
Interactions between effectors and their host targets are often weak or transient, making them difficult to identify. We describe a protocol for covalent capture of effector substrates in living cells using genetic code expansion technology. The effector-substrate complexes are captured by the crosslinker and subsequently purified with tandem chromatography. We detail steps for mass spectrum analysis and substrate verification. While the steps here are specific for substrates of enteropathogenic E. coli in HEK293T cells, the protocol has broader applications. For complete details on the use and execution of this protocol, please refer to Li et al. (2021). Covalent capture using site-specific, incorporated photo-crosslinker Identify weak or transient interactions of pathogens in living host cells Steps for mass spectrometry analysis and substrate verification Applicable for identifying interaction partners of proteins other than effectors Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Interactions between effectors and their host targets are often weak or transient, making them difficult to identify. We describe a protocol for covalent capture of effector substrates in living cells using genetic code expansion technology. The effector-substrate complexes are captured by the crosslinker and subsequently purified with tandem chromatography. We detail steps for mass spectrum analysis and substrate verification. While the steps here are specific for substrates of enteropathogenic E. coli in HEK293T cells, the protocol has broader applications.
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影响因子：
14.8
作者：
通讯作者：
--
基因整合的交联剂揭示了耐酸性中的分子伴侣合作。
影响因子：
14.8
作者：
Zhang, Meng;Lin, Shixian;Chen, Peng R.
通讯作者：
Chen, Peng R.
影响因子：
3.2
作者：
Seidel, Lisa;Zarzycka, Barbara;Coin, Irene
通讯作者：
Coin, Irene
DOI：
10.1073/pnas.0234824100
发表时间：
2003-01-07
影响因子：
11.1
作者：
Wang, L;Zhang, ZW;Schultz, PG
通讯作者：
Schultz, PG
影响因子：
64.5
作者：
Coin I;Katritch V;Sun T;Xiang Z;Siu FY;Beyermann M;Stevens RC;Wang L
通讯作者：
Wang L