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

Fu Zheng, Chenxin Yu, Xinyue Zhou and Peng Zou ()
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Fu Zheng: Peking University
Chenxin Yu: Peking University
Xinyue Zhou: Peking University
Peng Zou: Peking University

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-38565-8

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