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

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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Zheng Fu  

LAB HEAD: Peng Zou

PROVIDER: PXD037678 | Pride | 2023-07-20

REPOSITORIES: Pride