To decipher the spatiotemporally organized cellular proteome, we have integrated bioorthogonal decaging chemistry, spatial-temporal omics, and in-cell protein interaction mapping into a unified chemical biology platform. This approach allows us to probe living systems with unprecedented precision. Through genetically encoded and chemistry-driven activation or labeling strategies, we can rapidly modulate protein function, map proteomic and transcriptomic changes within specific subcellular niches, and capture dynamic cell–cell communication. In parallel, our photocrosslinking-based tools enable residue-level interrogation of protein–protein interactions and chromatin-associated networks directly inside living cells. Together, these complementary technologies provide a powerful framework for dissecting cellular organization, signaling, and molecular communication in real time, offering broadly applicable methods for understanding complex biological processes in their native context.
Recent representative work:
1) Bioorthogonal Decaging-based Proteomics
Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation (‘pulse’) of a kinase of interest, as well as global and in-depth characterization (‘chase’) of the perturbed proteome under living conditions. From this biological standpoint, we have been systematically applying our tools to activate and study specific kinases in living cells and addressing therapeutic needs. First, by genetically caging-and-decaging the catalytic lysine residue in protein kinases, we created a mechanism-based kinase activation strategy that is generally applicable for virtually any kinase of interest (ACS Cent. Sci. 2016). By coupling with ultradeep phosphoproteomics, we have “pulse-chased” the native substrates of various tyrosine kinases in living cells. This time-resolved approach is particularly powerful for deciphering the rewired signaling networks of oncogenic kinases. For example, we found that the major ubiquitin ligase-UBA1 is a substrate of the oncogenic mutant Src kinase. Phosphorylation of Y55 on UBA1 by this cancer-driving kinase decreased its ubiquitination activity with aberrant accumulation of malignant proteins that will ultimately lead to tumorigenesis (Nat. Chem. Biol. 2024). We also expanded this strategy to decipher the spatial-temporally organized phosphoproteome. The subcellular phosphoproteome characterization strategy-SubMAPP has allowed the monitoring of ER and mitochondria phosphoproteome in live cells and neurons (PNAS, 2021).
2）Bioorthogonal Decaging-based Spatial-temporal Omics
Chemical-based spatial-temporal omics technologies offer nongenetic tools for the in situ mapping of subcellular proteomes and transcriptomes in living systems. These approaches enable efficient protein or RNA labeling (Nat. Chem. 2025) within challenging biological contexts—including mitochondria (JACS, 2021), lysosomes (Nat. Catal. , 2025), the endoplasmic reticulum (PNAS, 2025), immune cells (JACS, 2024), primary live specimens (JACS, 2025), and living organisms (Nat. Commun. , 2024). They reveal previously hidden organelle constituents, capture dynamic cellular responses such as stress and immune activation, and support high-specificity, multi-omics integration across diverse cell types. Beyond subcellular profiling, these methodologies have been successfully extended to capture cell-cell interactions. Guided by this principle, we have further developed a suite of photocatalytic labeling technologies specifically designed for mapping cellular interactomes (JACS, 2024). These advances have inspired and led numerous studies in the field of tumor immunology, enabling in vivo, in situ decoding of immune processes and significantly advancing our understanding of cellular communication in complex biological systems. Collectively, these methods provide a powerful and evolving framework for interrogating cellular and tissue biology at a resolution and in environments previously inaccessible to conventional approaches.
3) Protein-protein interactions
Protein-protein interactions are of great significance for the regulation of cellular physiological activities. However, once a protein is isolated and purified, its original biological function is often lost and the corresponding protein-protein interactions and their functions are not able to be studied in vitro. Therefore, it is of great advantage to develop labeling techniques for protein interactions applicable to living systems. We have developed several highly specific protein photocrosslinking probes by combining genetic code expansion technology with photo crosslinking probes. The system is currently one of the most efficient and specific protein interactions capture tools available, and has been acquired by laboratories at nearly half a dozen universities or research institutions worldwide for the study of widespread protein-protein interactions in cells. We have leveraged these photocrosslinking probes to invent a “single-site-resolved multi-omics strategy” (SiTomics) that can capture the interacting gene loci and partner proteins of site-specific chromatin modifications simultaneously. This strategy was published in Cell, which allowed the linking of chromatinized proteome and genome that directly revealed how a specific histone modification functions in the spatially-organized chromatin architecture inside cells (Cell, 2023). We have also expanded this strategy to a Condensation-enhanced Photo-crosslinking (DenseMAP) technology depending on a metabolically incorporated photoaffinity amino acid, that allowed the spatial-temporally resolved dissection of protein interactome within bio-condensates inside living cells (Nat. Chem. 2025).
Representative publications：
1. Qin F*, Li B, Wang H, Ma S, Li J, Liu S, Kong L, Zheng H, Zhu R, Han Y, Yang M, Li K, Ji X*, Chen P*. “Linking chromatin acylation mark-defined proteome and genome in living cells”, Cell, (2023), 186, 1066-85
2. Li K, Xie X, Gao R, Chen Z, Yang M, Wen Z, Weng Y, Fan X, Zhang G, Liu L, Zeng X, Han Y, Cao M, Wang X, Li J, Yang Z, Li T, Chen P*. “Spatiotemporal protein interactome profiling through condensation-enhanced photocrosslinking”, Nat. Chem. (2025), 17, 111–123.
3. Bi Y, Yu L, Deng Q, Kong L, Guo F, Zhang Y, Wang R, Chen P* Liu J,* Fan X*, “Photocatalytic Proximity Labeling-Enabled Subcellular-Resolved RNA Profiling and Synchronous Multi-omics Investigation.” Nat. Chem. (2025), 17, 1871–1882
4. Zhang Y, Liu Z, Zhou N, Guo F, Guo H, Chen X, Chen P*, Fan X*, “In situ Lysosomal Proteomics Enabled by Bioorthogonal Photocatalytic Proximity Labeling.” Nat. Catal. (2025), 162-77
5. Zhou N, Zhang Y, Chen P*, Fan X*, “Time-Resolved Photocatalytic Proximity Labeling Uncovers ER Proteome Dynamics Underlying UPR to Apoptosis Transition.” Proc. Natl. Acad. Sci. U.S.A. 2025, 122 (32) e2503115122
6. Lou Z, Zhang Y, Liang X, Cao M, Ma Y, Chen P*, Fan X*, “Deep-Red and Ultrafast Photocatalytic Proximity Labeling Empowered in situ Dissection of Tumor-Immune Interactions in Primary Tissues.” J. Am. Chem. Soc. 2025, 9716-26
7. ‘Liu, Z.#; Guo, F.#; Zhu, Y.; Qin, S.; Hou, Y.; Lin, F.; Guo, H.; Chen P*; Fan X.*, Bioorthogonal photocatalytic proximity labeling in primary living samples. Nat. Commun. (2024) 15, 2712
8. Huang Z, Liu Z, Xie X, Zeng R, Chen Z, Kong L,Fan X*,Chen P*. “Bioorthogonal photocatalytic decaging-enabled mitochondrial proteomics”, J. Am. Chem. Soc. 2021, 143, 18714-20
9. Liu, Y.; Zeng, R.; Wang, R.; Weng, Y.; Wang, R.; Zou, P.; Chen P*, Spatiotemporally resolved subcellular phosphoproteomics. Proc. Natl. Acad. Sci. USA. 2021, 118 (25), e2025299118.
10. He D, Xie X, Yang F, Zhang H, Su H, Ge Y, Song H, Chen P* “Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker”, Angew. Chem. Int. Ed. 2017, 56, 14521-5.
11. Xie X, Li X, Qin F, Lin J, Zhang G, Zhao J, Bao X, Zhu R, Song H, Li X, Chen P* “Genetically encoded photoaffinity histone marks”, J. Am. Chem. Soc. 2017, 139, 6522-5.
12. Yang Y, Song H, He D, Zhang S, Dai S, Xie X, Lin S, Hao Z, Zheng H, Chen P*“Genetically encoded releasable photocrosslinking strategies for studying protein-protein interactions in living cells”, Nat. Protocol. (2017), 12, 2147-68.
