2. Academic Validation
  3. Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

  • Nat Commun. 2023 May 23;14(1):2978. doi: 10.1038/s41467-023-38565-8.
Fu Zheng 1,  Chenxin Yu 2 3,  Xinyue Zhou 2,  Peng Zou 4 5 6 7
Affiliations

Affiliations

  • 1 College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871, China.
  • 2 Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.
  • 3 College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China.
  • 4 College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871, China. zoupeng@pku.edu.cn.
  • 5 Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China. zoupeng@pku.edu.cn.
  • 6 PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, 100871, China. zoupeng@pku.edu.cn.
  • 7 Chinese Institute for Brain Research (CIBR), Beijing, 102206, China. zoupeng@pku.edu.cn.
PMID: 37221179 DOI: 10.1038/s41467-023-38565-8
Abstract

Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a Reactive Oxygen Species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 477 mitochondrial proteins with 94% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum (ER). The temporal control of RinID enables pulse-chase labeling of ER proteome in HeLa cells, which reveals substantially higher clearance rate for secreted proteins than ER resident proteins.

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