In situ activity-based protein profiling of serine hydrolases in E. coli

• We describe a method for in cell activity based protein profiling.
• The method is applied to E. coli.
• We compare the efficiency and patterns of labeling in cell vs in lysate.
• Mainly qualitative differences are noted.
• We perform mass spectrometry to identify the labeled proteins.

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.

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