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

Abstract

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.