Supporting data for "VISIBLE LIGHT-ACTIVATED COVALENT LABELING PROBES ENABLE SPATIOTEMPORAL MAPPING OF LOCALIZED PROTEIN ENVIRONMENTS"

All the data below these files are about the mitochondrial proteomic analysis by using our probes, particularly for HeLa cells. The data include the procedures of the mitochondrial proteomics and the RAW data presented by Excel. The data was proceeded in Excel. We developed the first red-light-induced covalent crosslinking strategy for endogenous protein labeling in living cells that bypasses additional photocatalysts or photosensitizers. This files present the first MIM-localized small-molecule probes for mitochondrial proteomic analysis, particularly by using RAW cells under LPS stimulation. The data was proceeded in Excel, GOCC, and metascape. By using RAW cells as models, we proved that our approach circumvents genetic manipulation, exhibits minimal cytotoxicity, and offers a versatile tool for studying mitochondrial proteomics in live cells, advancing our understanding of mitochondrial adaptations in health and disease.Chapter 2: we developed two red-shifted coumarin-scaffold fluorescent probes that bypass photocatalysts, directly generating reactive carbene intermediates under green or red LED light irradiation for covalent protein labeling. Both probes exhibited efficient labeling of bovine serum albumin (BSA) without amino acid preference, including for leucine residues.Chapter 3: Probes derived from a carbonic anhydrase (CA) inhibitor and red light-activated pyridinium-diazo-coumarin successfully labeled CA-II in vitro upon red LED light irradiation. However, detection of endogenous CAs using our probes was unsuccessful. Similarly, probes derived from bromodomain-containing protein (BRD) inhibitors and pyridinium-diazo-coumarin failed to detect endogenous BRDs, likely due to insufficient ligand affinity or poor intracellular accessibility.Chapter 4: Notably, through hydrophobicity optimization, our red light-activated pyridinium-diazo-coumarin probes enabled selective, high-efficiency labeling of the mitochondrial inner membrane (MIM) across diverse cell lines. Applications in HeLa cells, hypoxia-stimulated HEK293T cells, and LPS-activated RAW264.7 macrophages further confirmed their utility in capturing mitochondrial proteomics, particularly MIM proteomic dynamics under physiological conditions.<br>