Revealing the Hydrophobic Interactions between Annular Lipids and Transmembrane Peptides via Photo-Tagging and Mass Spectrometry

Annular lipids, which constitute the lipid layer directly interacting with protein transmembrane domains, are essential for maintaining protein structure and function. Nevertheless, the analysis of the dynamic and weak noncovalent interactions between annular lipids and proteins remains challenging, leaving their interaction details largely underexplored. To address this challenge, we have developed novel phototagging probes, NHS-c-Bpa and IAM-c-Bpa, to capture these weak interactions in peptide-liposome models. The probes feature an NHS moiety for lysine derivatization or an iodoacetamidyl group for cysteine conjugation, a basic pH-cleavable linker, and a p -benzoyl- l -phenylalanine (Bpa) group for tagging annular lipids upon UV irradiation. The cross-linked peptide-lipid products are subjected to saponification, followed by liquid chromatography-tandem mass spectrometry (MS/MS) analysis. Importantly, MS/MS analysis resolves product isomers that differ only in the tagging positions along the fatty acyl chains, enabling the construction of tagging profiles and the determination of the penetration depth of the targeted amino acid residues within the lipid bilayer. Combined with all-atom molecular dynamics simulations, we reveal the structural details of hydrophobic mismatches induced by model transmembrane peptides and determine the identity and stoichiometry of annular lipids surrounding individual peptides. Although currently demonstrated with peptide models, our method can be further developed to investigate annular lipid–protein interactions, providing critical experimental insights that are not attainable through existing structural biology techniques.