Microenvironment-Specific Enrichment Strategy Enables Lysosomal Proteomic Dynamics Analysis

Lysosomes are vital organelles for degradation, recycling, and cellular homeostasis, impacting signaling and metabolism. Analyzing the lysosomal proteome dynamics is key to understanding these roles, but the acidic environment and low abundance of lysosomes make proteomic analysis challenging. Herein, we developed a lysosome-localizable reactive diazirine molecule MDA and demonstrated its enhanced labeling capability in the lysosomal microenvironment. Furthermore, we introduced a novel microenvironment-specific enrichment (MiSE) strategy for profiling the lysosomal proteome, combining MDA-based labeling with affinity enrichment. We successfully applied MiSE to profile the lysosomal proteome in living SH-SY5Y cells, achieving coverage of 132 lysosome-annotated proteins. Moreover, by coupling MiSE with data-independent acquisition (DIA) analysis, we explored dynamic changes in the lysosomal proteome upon inhibition of the ubiquitin-proteasome system using four proteasome inhibitors. Our results reveal 117 UPS-inhibition-related lysosomal proteins, highlighting their involvement in stress response and cell cycle regulation. Notably, we observe distinct proteomic signatures for each inhibitor, suggesting unique mechanisms of lysosomal response to UPS inhibition. Therefore, MiSE offers a powerful tool for investigating the dynamic lysosomal proteome, providing insights into cellular homeostasis and disease pathogenesis. This approach holds significant potential for advancing the understanding of lysosomal function and developing novel therapeutic strategies.