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Activity‐based protein profiling (ABPP) is a chemical proteomic method for investigating functional states of proteins in native biological settings. By quantifying changes in probe binding states of active and regulatory protein sites, ABPP reveals functional information on protein regulation and can be configured in competitive settings to determine global selectivity profiles of tool compounds and drugs in lysates, cells, and animals. Chemical probes used for ABPP analyses can target protein families with conserved enzymatic or structural features or can broadly profile the proteome using electrophiles with reactivity towards functional groups on amino acid side chains. The latter approach has provided insights to protein sites involved in allosteric regulation and non‐enzymatic functions. This review introduces quantitative ABPP workflows and discusses electrophilic groups used for ABPP profiling of functional sites in the proteome with an emphasis on tyrosine residues.

Diacylglycerol lipase-beta (DAGLβ) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLβ ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLβ in primary macrophages leads to LKB1–AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLβ blockade, thereby directly supporting DAGLβ–AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.

We provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.