Cell Surface Deazaflavin-Diazirine Energy-Transfer (DarT) Photoproximity Labeling Using Antibody Conjugates.

Elucidating protein-protein interactions plays a crucial part in understanding disease mechanisms and advancing pharmacological research. Photocatalytic proximity labeling using antibody-catalyst conjugates enables the highly target-specific analysis of protein-protein interactions on the cell surface without altering the native cellular state through genetic manipulation. Here, we describe an extension of the deazaflavin-diazirine energy-transfer (DarT) labeling platform through the development and evaluation of trastuzumab-deazaflavin (Tra-dFl) conjugates for mapping the extracellular microenvironment of human epidermal growth factor receptor 2 (HER2). Four Tra-dFl conjugates were synthesized via azide-DBCO click chemistry, varying in PEG linker size and catalyst loading: Tra-PEG0-dFl, Tra-PEG6-dFl, Tra-PEG12-dFl, and Tra-bis-dFl, exhibiting a branched linker for dual attachment. Imaging and proteomic pulldown experiments revealed that linker size influences biotinylation efficiency and proteomic enrichment, resulting in Tra-PEG12-dFl emerging as the most effective construct, enabling the enrichment of cancer-associated cell surface proteins in HER2-positive SK-BR-3 cells. Evaluation of catalyst valency using a branched linker resulted in fewer enriched proteins, suggesting that linker architecture is a more critical parameter for conjugate performance than increased catalyst loading. Together, these findings provide guidelines for antibody-based deazaflavin conjugates and expand the applicability of DarT labeling for target-directed surfaceome mapping.