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

Fu Zheng, Chenxin Yu, Xinyue Zhou, View ORCID ProfilePeng Zou
doi: https://doi.org/10.1101/2022.08.01.502286
Fu Zheng
aCollege 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
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Chenxin Yu
bAcademy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
eCollege of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China
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Xinyue Zhou
bAcademy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
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Peng Zou
aCollege 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
bAcademy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
cPKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, 100871, China
dChinese Institute for Brain Research (CIBR), Beijing 102206, China
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  • ORCID record for Peng Zou
  • For correspondence: zoupeng@pku.edu.cn
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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 394 mitochondrial proteins with 97% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted August 02, 2022.
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Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome
Fu Zheng, Chenxin Yu, Xinyue Zhou, Peng Zou
bioRxiv 2022.08.01.502286; doi: https://doi.org/10.1101/2022.08.01.502286
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Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome
Fu Zheng, Chenxin Yu, Xinyue Zhou, Peng Zou
bioRxiv 2022.08.01.502286; doi: https://doi.org/10.1101/2022.08.01.502286

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