Genome sequencing projects have revolutionized our view of the complexity of prokaryotic and eukaryotic proteomes, however, we are also left with a daunting challenge of functionally annotating these large number of predicted proteins. Experimental chemoproteomic methods, such as activity-based protein profiling (ABPP), have been developed aiming at systematically discovering new functional targets directly from native proteomes. Our research group have developed various ABPP-based strategies to uncover sites of ligand binding, post-translational modifications (PTMs) as well as enzyme catalysis in living cells and tissues. In this talk, I will present some of our recent work in profiling sites of PTMs by a functionally intriguing metabolite named “itaconate”, which has been with strong immunoregulatory and antibacterial activity. Our chemical proteomics studies have revealed a large number of novel and interesting sites of “itaconation” from host macrophages, and provided important clues for further functional characterization of itaconate in mediating host-pathogen interactions.