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<p>Dynamic protein complex assembly is critical for regulating various biological processes. Proximity labeling (PL), best represented by the ascorbate peroxidase APEX2, allows these molecular events to be captured in living cells in a spatiotemporal manner. However, the hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) dependence of APEX2 has hindered its application in sensitive living systems. Here we introduce ROProx, a radical- and oxygen-driven photoreactive PL technology that leverages the chemically evolved biotin-naphthylamine probe BN2, which has strong binding affinity for APEX2, and the unexpected tyrosyl radicals in APEX2. ROProx labels dynamic cytosolic protein complexes in living cells within seconds, with a range of 10 nm, and is precisely controlled by mild blue light irradiation without H<sub>2</sub>O<sub>2</sub>. Additionally, we apply ROProx to explore the phosphotyrosine-dependent GRB2 interactome in living mice by simply injecting BN2 for 5 minutes. ROProx should, therefore, open broad opportunities for PL chemical evolution and applications in other living systems.</p><p></p>

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H2O2-free proximity proteomics for exploring dynamic protein complexes in living systems

  • 期刊论文

  • 发表日期：

    2026年5月28日

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  • Mi Ke,
  • Fuchao Liang,
  • Guangqin Wang,
  • An He,
  • Baixu Ma,
  • Zheyi Liu,
  • Jie Wang,
  • Yan Xu,
  • Yanli Li,
  • Zhe Dong,
  • Zhihua Zhong,
  • Zhiyao Tang,
  • Pengyu Sun,
  • Kangmin He,
  • Fangjun Wang,
  • Zhiyong Liu,
  • Xueming Yang,
  • Lizhi Tao,
  • Ruijun Tian

摘要

Dynamic protein complex assembly is critical for regulating various biological processes. Proximity labeling (PL), best represented by the ascorbate peroxidase APEX2, allows these molecular events to be captured in living cells in a spatiotemporal manner. However, the hydrogen peroxide (H2O2) dependence of APEX2 has hindered its application in sensitive living systems. Here we introduce ROProx, a radical- and oxygen-driven photoreactive PL technology that leverages the chemically evolved biotin-naphthylamine probe BN2, which has strong binding affinity for APEX2, and the unexpected tyrosyl radicals in APEX2. ROProx labels dynamic cytosolic protein complexes in living cells within seconds, with a range of 10 nm, and is precisely controlled by mild blue light irradiation without H2O2. Additionally, we apply ROProx to explore the phosphotyrosine-dependent GRB2 interactome in living mice by simply injecting BN2 for 5 minutes. ROProx should, therefore, open broad opportunities for PL chemical evolution and applications in other living systems.