Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification

Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. Recently, we reported on the establishment, by ABPP, of the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Here, we describe a protocol for identifying the in vivo selectivity profile of covalent inhibitors using ABPP with label-free quantitative proteomics as exemplified by experimental data on the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The protocol has been optimized using label-free quantification methods to have high proteome coverage and to allow the simultaneous comparison of multiple biological samples. The stages of the protocol include tissue lysis, probe incubation, enrichment, sample preparation, liquid chromatography-mass spectrometry measurement, processing and data analysis. The results include evidence of target engagement in a native proteome and the identification of potential off-targets for the inhibitor under investigation. The entire protocol takes at least four days (depending on the number of samples).