Future biological research must carefully consider characterizing functional proteins and pathways within the native context in which they exist. Cell lysis results in dramatic perturbations to various metabolic pathways while disrupting the dynamic subcellular spatial and temporal interactions. We developed a chemical probe that targets select functionally active enzymes localized within a specific organelle, the lysosome. To do so, we developed a multimodal activity-based probe for targeting lysosomes, which have an acidic internal pH, for measuring subcellular native enzymatic activity via super-resolution structured illumination microscopy (SIM) and LC-MS. A cathepsin-reactive warhead conjugated to a weakly basic amine and a click-chemistry compatible alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by fluorescence microscopy in J774 mouse macrophage cells. The accumulation occurred within rapid timescales (~5 min). Lysosome accumulation was confirmed by overlaying fluorescent organelle marker antibodies with probe-labeling fluorescence using confocal laser microscopy and SIM. Analysis of in vivo labeled J774 cells by LC-MS identified high selectivity for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, demonstrated a large increase in the number of tagged proteins and an increase in cathepsin activity. This result is consistent with expectations as autophagy induces cell death and organelle destruction leading to increased lysosomal content. Organelle-targeting ABPP holds great promise for the characterization of enzyme activities in the myriad of diseases linked to specific subcellular locations. Our research is ongoing in this area.

Organelle-specific activity-based protein profiling in live macrophage cells revealed by super-resolution imaging and proteomics

248th National Meeting (2014)

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