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. 2020 Sep 15;1(3):100109.
doi: 10.1016/j.xpro.2020.100109. eCollection 2020 Dec 18.

Protocol for Site-Specific Photo-Crosslinking Proteomics to Identify Protein-Protein Interactions in Mammalian Cells

Chengjie Chen  1 , Tao Peng  1
Affiliations

Protocol for Site-Specific Photo-Crosslinking Proteomics to Identify Protein-Protein Interactions in Mammalian Cells

Chengjie Chen et al. STAR Protoc. .

Abstract

Protein-protein interactions (PPIs) play essential roles in almost all aspects of cellular processes. However, PPIs remain challenging to study due to their substoichiometry, low affinity, dynamic nature, and context dependence. Here, we present a protocol for the capture and identification of PPIs in live mammalian cells, which relies on site-specific photo-crosslinking in live cells, affinity purification, and quantitative proteomics. The protocol facilitates efficient and reliable identification of the interacting proteins of a given protein of interest in live cells. For complete details on the use and execution of this protocol, please refer to Wu et al. (2020).

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Workflow for Site-Specific Photo-Crosslinking Proteomics to Identify PPIs in Mammalian Cells (A) Schematic for evaluating site-specific incorporation of DiZPK into POI in mammalian cells. (B) Schematic for examining photo-crosslinking of DiZPK-modified POI in mammalian cells. (C) Schematic for profiling and validating POI-interacting proteins with site-specific photo-crosslinking in mammalian cells.
Figure 2
Figure 2
Results for Site-Specific Photo-Crosslinking of IFITM3 in HEK293T Cells (A) Western blot analysis of HEK293T cells expressing HA-IFITM3-TAG mutants in the presence or absence of DiZPK. (B) Western blot analysis of photo-crosslinking complexes in HEK293T cells expressing HA-IFITM3-TAG mutants with or without UV irradiation. (C) Quantitative proteomic analysis for identification of photo-crosslinked IFITM3-interacting proteins. VCP, highly enriched in photo-crosslinking samples, is marked in red. (D) Western blot analysis for validation of IFITM3-VCP photo-crosslinking complex. The red asterisk and double asterisks indicate co-immunoprecipitated VCP and photo-crosslinked IFITM3-VCP complex, respectively. Note that N-terminally HA-tagged IFITM3 constructs were used in these experiments.
Figure 3
Figure 3
Experimental Design of the Quantitative SILAC Proteomic Analysis (A) Schematic for forward SILAC experiment. Heavy SILAC cells are UV-irradiated, while light SILAC cells are not UV-irradiated. (B) Schematic for forward SILAC experiment. Light SILAC cells are UV-irradiated, while heavy SILAC cells are not UV-irradiated.
See this image and copyright information in PMC

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