Photocatalytic proximity labeling coupled with cross-linking for deciphering of the lysosome proteome in living cells

Background

Lysosomes are essential organelles that play vital roles in the degradation and recycling of a variety of biomolecules. 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 struggles 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 dynamic lysosomal proteome and revealed stress-induced metabolic alterations.

Significance

The developed strategy enhanced lysosomal proteomic coverage via a high-precision integration platform, allowing systematic identification of both membrane-associated and luminal proteins within lysosome, thereby providing mechanistic insights into subcellular biology and unlocking new frontiers in cellular research.