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Photocatalytic Proximity Labeling Coupled with Cross-linking for Deciphering of the Lysosome Proteome in Living Cells-new

ABSTRACT: Background: Lysosomes are essential organelles that play vital roles in the degradation and recycling of a variety of bio-mole-cules. To fully understand their molecular mechanisms, high spatiotemporal identification of the lysosome proteome in living cells is critical. Although photocatalytic proximity labeling is a powerful tool for profiling subcellular proteome, it often strug-gles with low labeling efficiency, particularly for low-abundance proteins. Results: To overcome this, we introduced a novel strategy that integrates photocatalytic proximity labeling with cross-linking. The lysosome-targeting photosensitizer (MP-AcDBF) catalyzed the covalent reaction between proximal proteins and enrichable nucleophilic substrate within lysosome based on singlet oxygen. Then the cross-linker allowed difficult-to-label proteins (low-abundance or transiently localized proteins) to be linked to pre-labeled ones, thereby expanding the scope of protein identification while preserving lysosomal specificity via enrichment of labeled handles. Using the cross-linking-assisted photocatalytic proximity labeling (CLAPL) strategy, we successfully identified 238 lysosome-annotated proteins in living HeLa cells, covering luminal, transmembrane and membrane-associated proteins, enabling a relative increase compared to method without cross-linking (238 vs 197). Additionally, CLAPL was further applied to map dynam-ic lysosomal proteome and revealed stress-induced metabolic alterations.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lihua Zhang

PROVIDER: PXD068636 | iProX | Fri Sep 19 00:00:00 BST 2025

REPOSITORIES: iProX

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Photocatalytic Proximity Labeling Coupled with Cross-linking for Deciphering of the Lysosome Proteome in Living Cells

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