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 Visible-light-controlled Lysine-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 MS data analysis—but also reveals dynamic interactomes in various scenarios. The VL-XL strategy successfully profiles the time-resolved, EGF-stimulated 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 SESN2 and opening an innovative avenue for identifying novel targets for degradation. Overall, the VL-XL strategy offers a robust chemical tool for decoding protein complexes and dynamic interactomes, inspiring innovative solutions to address unresolved questions in proteomics, systems biology and drug discovery.

中文翻译：

---

可见光控制的赖氨酸选择联可解码活细胞中的蛋白质复合物和动态相互作用组

交联策略已成为破译蛋白质复合物和蛋白质-蛋白质相互作用 （PPI） 的一种有吸引力的技术。然而，常用的交联策略对天然生物环境中蛋白质复合物和动态 PPI 的精确分析提出了重大挑战。在这里，我们报告了第一个可见光诱导赖氨酸特异性同型双功能光交联剂的开发，并介绍了可见光控制的赖氨酸选择联 （VL-XL） 策略，用于深入分析蛋白质复合物和分析活细胞中的动态相互作用组。通过协同整合时间控制、高生物相容性和赖氨酸选择性的优势，VL-XL 策略不仅为蛋白质复合物研究提供了有效的解决方案——实现残基特异联肽，提供高可信度数据和简化的 MS 数据分析——而且还揭示了各种情况下的动态相互作用组。VL-XL 策略成功地分析了时间分辨的 EGF 刺激的 EGFR 相互作用组，为 EGFR 信号转导的调节机制提供了有价值的见解。更重要的是，VL-XL 策略有效地揭示了分子胶降解剂诱导的 E3 连接酶相互作用组，从而发现了 SESN2 等新底物，并为识别新的降解靶点开辟了一条创新途径。总体而言，VL-XL 策略为解码蛋白质复合物和动态相互作用组提供了强大的化学工具，激发了创新解决方案来解决蛋白质组学、系统生物学和药物发现中未解决的问题。