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Proximity labeling of proteome and metabolome at organelle membranes with split-PLD reveals hydrogen sulfide-inhibited cuproptosis

ABSTRACT: Crosslinking mass spectrometry has been widely applied to probe protein–protein interactions. However, proximity labeling at organelle membranes, particularly beyond proteins, is still poorly characterized. Here, we develop a splitted phospholipase D (split-PLD)–based proximity labeling strategy targeted to mitochondrial and endoplasmic reticulum (ER) membranes, enabling the identification of substantially more membrane-associated proteins than existing methods such as TurboID and APEX2, and revealing proximal proteome atlas with different distances from organelle membranes, including functional assemblies. Importantly, integration of the split-PLD-driven labeling mechanism with click chemistry allows interrogation of proximal reactions between metabolites and lipids at mitochondrial and ER membranes without organelle isolation. Using this platform, we identified a gas molecule hydrogen sulfide (H2S) at mitochondrial membrane interfaces and demonstrate that H2S suppresses cuproptosis in the presence of copper ionophore elesclomol. Collectively, we establish a versatile platform for interrogating molecular activities at organellar membranes, further conceptualizing “contactomics”.

ORGANISM(S): Homo Sapiens

SUBMITTER: Yaoyang Zhang

PROVIDER: PXD073432 | iProX | Thu Jan 22 00:00:00 GMT 2026

REPOSITORIES: iProX

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