Deazaflavin-Diazirine Energy-Transfer (DarT) Photoproximity Labelling of the HER2 Interactome Using Antibody Conjugates

ABSTRACT: Elucidating protein-protein interactions plays a crucial part in understanding disease mecha-nisms and advancing pharmacological research. Photocatalytic proximity labelling using anti-body–catalyst conjugates enables the highly target-specific analysis of protein-protein inter-actions on the cell surface without altering the native cellular state through genetic manipu-lation. Here, we describe an extension of the deazaflavin–diazirine energy-transfer (DarT) labelling 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 synthesised 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 effi-ciency and proteomic enrichment, resulting in Tra–PEG12–dFl to emerge as the most effec-tive construct, enabling 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 more critical for conjugate perfor-mance than increased catalyst loading. Together, these findings provide guidelines for anti-body-based deazaflavin conjugates and expand the applicability of DarT labelling for target-directed surfaceome mapping.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Max Ruwolt  

LAB HEAD: Christian Hackenberger

PROVIDER: PXD076830 | Pride | 2026-08-04

REPOSITORIES: Pride