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Title: Organelle-Specific Activity-Based Protein Profiling in Living Cells

A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.
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Resource Type:
Journal Article
Resource Relation:
Journal Name: Angewandte Chemie International Edition, 53(11):2919-2922
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org:
Country of Publication:
United States
Environmental Molecular Sciences Laboratory