Covalent labeling of amino acids in proteins by reactive small molecules, in particular at cysteine SH and lysine NH groups, is a powerful approach to identify and characterize proteins and their functions. However, for the less-reactive carboxylic acids present in Asp and Glu, hardly any methodology is available. Employing the lipoprotein binding chaperone PDE6δ as an example, we demonstrate that incorporation of isoxazolium salts that resemble the structure and reactivity of Woodward's reagent K into protein ligands provides a novel method for selective covalent targeting of binding site carboxylic acids in whole proteomes. Covalent adduct formation occurs via rapid formation of enol esters and the covalent bond is stable even in the presence of strong nucleophiles. This new method promises to open up hitherto unexplored opportunities for chemical biology research. © 2017 Elsevier Ltd