Visible‐Light‐Controlled Lysine‐Selective Crosslinking Decodes Protein Complexes and Dynamic Interactomes in Live Cells

Abstract

Abstract Crosslinking strategies have emerged as an attractive technology for deciphering protein complexes and protein–protein interactions (PPIs). However, commonly used crosslinking strategies present significant challenges for the precise analysis of protein complexes and dynamic PPIs in native biological environments. Here, we report the development of the first visible‐light‐inducible lysine‐specific homobifunctional photo‐crosslinkers and introduce V isible‐light‐controlled L ysine‐selective crosslinking (VL‐XL) strategy for in‐depth analysis of protein complexes and profiling dynamic interactomes in live cells. By synergistically integrating the advantages of temporal control, high biocompatibility, and lysine selectivity, the VL‐XL strategy not only provides an effective solution for protein complexes studies—achieving residue‐specific crosslinked peptides, delivering high‐confidence data and streamlined mass spectrometry (MS) data analysis—but also reveals dynamic interactomes in various scenarios. The VL‐XL strategy successfully profiles the time‐resolved, epidermal growth factor (EGF)‐stimulated epidermal growth factor receptor (EGFR) interactome, providing valuable insights into regulatory mechanisms of EGFR signaling. More importantly, the VL‐XL strategy effectively unveils molecular glue degrader‐induced E3 ligase interactome, leading to discovery of neo‐substrates such as Sestrin‐2 (SESN2) and opening an innovative avenue for identifying novel targets for degradation. Overall, the VL‐XL strategy provides a robust chemical tool, inspiring innovative solutions to address unresolved questions in multiple fields.