In situ lysosomal proteomics enabled by bioorthogonal photocatalytic proximity labelling

Abstract

In situ deciphering of lysosome proteomes is crucial for understanding cellular processes and diseases, but is challenging due to its digestive, acidic environment that renders proximity labelling enzymes incompatible. Here we have developed a photocatalytic proximity labelling technique, CAT-Lyso, for in situ lysosomal proteomics. By employing a lysosome-targeting photocatalyst/thioquinone methide labelling probe pair, CAT-Lyso enables the generation of a reactive thioquinone methide intermediate via photoredox catalysis, facilitating efficient lysosomal proteome labelling in diverse cell lines, including hard-to-transfect macrophages (RAW264.7) and B lymphocytes (Raji). CAT-Lyso successfully identified cell type-specific lysosomal proteomic patterns and uncovered previously unrecognized lysosomal proteins, such as SCAMP3, NAGPA, GLG1 and MFSD14B. Furthermore, CAT-Lyso enabled quantitative profiling of lysosomal proteome dynamics under perturbations such as rapamycin-mediated mTOR inhibition, revealing pronounced ferritinophagy that evokes a coordinated labile iron-resisting program in cancer cells. With its in situ labelling, non-genetic operation, high specificity and photocontrollability, CAT-Lyso provides a powerful tool for investigating lysosome proteome dynamics in living systems. In situ profiling of lysosomal proteomes is impeded by the acidic and digestive environment of lysosomes. Now, a bioorthogonal photocatalytic system (CAT-Lyso) is developed that is compatible with these conditions, enabling the proximity labelling of lysosomal proteomes within living cells.