A Protocol for Protein Profiling Using Chemoselective Cleavable Linker Probes in Semi-permeabilized Cells.

Activity-based protein profiling using activity-based probes (ABPs) resulted in the identification of various enzymes that are involved in the onset and progress of diseases. Detection of such proteins, often expressed at low abundance, is greatly enhanced by incorporating chemically cleavable linkers in the ABP of choice. Initial affinity purification, followed by tailored chemical cleavage of the linker, allows for specific release of the captured enzymes and their interaction partners. When the ABPs are delivered directly to semi-permeabilized cells, in contrast to a crude cell lysate, the sensitivity and efficacy of cell impermeable probes can be enhanced even further.