Combining Chemical Protein Synthesis and Cell Delivery for Photoproximity Labeling in Living Cells

Mapping a protein’s local environment is essential for understanding its cellular roles. Proximity labeling (PL) identifies nearby proteins, while photoproximity labeling (PPL) adds temporal control through light activation. However, intracellular applications of these methods commonly rely on genetically encoded fusions that could alter protein localization or function and offer limited control over defined, site-specific post-translational modifications (PTMs). Here, we combine chemical protein synthesis with bead loading (BL) to enable live-cell PPL using fully chemically synthesized photocatalyst–protein of interest (PC–POI) conjugates. Synthesis enables site-specific PC attachment, while BL delivers conjugates directly into cells, bypassing endocytosis and preventing endosomal trapping. As a proof of concept, we demonstrate the strategy using chemically synthesized small ubiquitin-like modifier (SUMO) proteins. Notably, PC selection was critical: conjugation to Eosin Y altered SUMO2 nuclear localization, whereas 5-deazaflavin (DF) preserved both nuclear localization and the ability to conjugate to cellular proteins. Using TAMRA–DF–SUMO conjugates, blue-light-triggered deazaflavin–diazirine energy-transfer (DarT) labeling captured nearby proteins for enrichment and quantitative proteomics. Comparing SUMO2 with its non-conjugatable variant SUMO2ΔG93 and its paralog SUMO1 showed that the approach resolves proximity profiles associated with conjugation competence and paralog identity. The biological relevance of the SUMO interactome was supported by established associations of selected proteins and by the finding that approximately 70% of proteins preferentially captured by SUMO2 over SUMO2ΔG93 were reported as SUMO2 substrates. This work establishes live-cell PPL without genetically encoded labeling fusions or target-protein overexpression, opening opportunities to investigate how defined, site-specific PTMs shape local protein environments.