We describe a method for in cell activity based protein profiling.
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The method is applied to E. coli.
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We compare the efficiency and patterns of labeling in cell vs in lysate.
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Mainly qualitative differences are noted.
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We perform mass spectrometry to identify the labeled proteins.
Abstract
A fluorophosphonate based alkyne activity probe was used for the selective labeling of active serine hydrolases in intact Escherichia coli cells. A biotin-azide tag was subsequently attached to the alkyne functionality of the probe with copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Comparison of proteins from in-cell and lysate labeled preparations suggested qualitatively similar patterns of reactivity in both preparations. Approximately 68%, 30 of the total 44 serine hydrolases detectable in E. coli were labeled with the probe indicating significant coverage with a single probe. The methods described here offer a useful tool for profiling and monitoring serine hydrolase activity in situ.
Graphical abstract
Abbreviations
ABPP
activity based protein profiling
SerHs
serine hydrolase
FP-probe
fluorophosphonate-based probe
THPTA
[tris(3-hydroxy-propyl-triazolyl-methyl)]amine
CuAAC
copper-catalyzed azide-alkyne cycloaddition
TAMRA
carboxytetramethylrhodamine
SDSPAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis