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  3. 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

  • STAR Protoc. 2022 Dec 16;3(4):101882. doi: 10.1016/j.xpro.2022.101882.
Pan Li 1,  Jingxiang Li 1,  Haiyan Ren 2
Affiliations

Affiliations

  • 1 Division of Respiratory and Critical Care Medicine, West China Hospital of Sichuan University, Chengdu 610041, China.
  • 2 Division of Respiratory and Critical Care Medicine, West China Hospital of Sichuan University, Chengdu 610041, China; Collaborative Innovation Center of Biotherapy, Chengdu 610041, China. Electronic address: [email protected].
PMID: 36595886 DOI: 10.1016/j.xpro.2022.101882
Abstract

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).1.

Keywords

Mass Spectrometry; Microbiology; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry.

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