Bifunctional Lipid–Protein Cross-linking Efficiency and Reaction Products

Bifunctional diazirine lipids are versatile tools for mapping protein-lipid interactions and cellular localization by photo-cross-linking. Yet, the cross-linking efficiency of these probes has not been systematically evaluated. We use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-cross-linking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein-lipid cross-links, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows.