Spatially confined photocatalytic proximity labeling via isothiocyanate chemistry enabled in situ surfaceome analysis

As a dynamic and functionally essential subset of the proteome, the surfaceome governs cell communication, signaling, and representing a major reservoir of therapeutic targets. Comprehensive and in-depth mapping of the surfaceome is indispensable for understanding the molecular basis of disease. Photosensitizer-mediated photocatalytic proximity labeling has emerged as a powerful tool for in situ surfaceome labeling with high spatiotemporal precision and without genetic manipulation. However, most photosensitizers depend on hydrophobic and electrostatic interactions, which are easily disrupted by membrane fluidity, leading to probe diffusion or endocytosis and ultimately limiting labeling efficiency.

To overcome this, we developed a spatially confined photocatalytic proximity labeling strategy that leveraged a novel positively charged isothiocyanate (SCN)-based photosensitizer (SCN-Py-AcDBF). The probe was initially adsorbed onto the cell surface via electrostatic interactions and subsequently restricted in its mobility via covalent anchoring to proteins through isothiocyanate chemistry. Upon photocatalytic activation, the probe generated ¹O₂ to induce covalent labeling of cell surface proteins and nearby nucleophilic substrates. Finally, we successfully identified 1124 CSPs from living HeLa cells with high quality, including 659 plasma membrane proteins, demonstrating improvement in coverage over methods without spatial confinement.

This SCN-based spatial confinement strategy significantly minimizes diffusion and prevents endocytosis of the photosensitizer, leading to enhanced labeling specificity and probe efficiency. This strategy improved both the accuracy and coverage of surfaceome profiling, providing a robust platform for comprehensive study of surfaceome dynamics and function.