Deciphering photosynthetic protein networks: a crosslinking‐ MS strategy for studying functional thylakoid membranes

SUMMARY Photosynthesis, which sustains life on Earth, depends on organized and yet adaptable protein assemblies embedded in specialized membranes known as thylakoids. Understanding how these complexes interact and reorganize within functional photosynthetic membranes is essential to reveal the molecular basis of energy conversion in cells. Here, we present an improved cross‐linking mass spectrometry strategy that captures native protein interactions in photosynthetically active thylakoid membranes from Arabidopsis thaliana and Spinacia oleracea . By monitoring photosynthetic performance during cross‐linking, we show that electron transport remains active, allowing structural analysis under physiological conditions. Moreover, we show that trimethylphenylammonium chloride as an adjuvant charged compound does not impair physiological activity, while boosting and diversifying cross‐link identifications. Mapping cross‐links onto known structures confirms the integrity of major photosynthetic complexes and uncovers previously uncharacterized assemblies involving regulatory and structural proteins. Integration with structural modeling and interaction network analysis identifies novel protein players within the photosynthetic machinery, providing molecular insights into their potential roles. This approach offers a broadly applicable framework for studying membrane protein organization and dynamics in functional bioenergetic systems.