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Angew Chem Int Ed Engl. 2014 Mar 10;53(11):2919-22. doi: 10.1002/anie.201309135. Epub 2014 Feb 6.

Organelle-specific activity-based protein profiling in living cells.

Author information

1
Biological Sciences Division, Pacific Northwest National Laboratory, 902 Battelle Blvd, Richland, WA 99352 (USA).

Abstract

A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin-reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.

KEYWORDS:

activity-based probes; fluorescence microscopy; lysosome; mass spectrometry; proteomics

PMID:
24505022
PMCID:
PMC4023867
DOI:
10.1002/anie.201309135
[Indexed for MEDLINE]
Free PMC Article

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