Nucleophilic amino acids make important contributions to protein function, including performing key roles in catalysis and serving as sites for post-translational modification. Electrophilic groups that target amino-acid nucleophiles have been used to create covalent ligands and drugs, but have, so far, been mainly limited to cysteine and serine. Here we report a chemical proteomic platform for the global and quantitative analysis of lysine residues in native biological systems. We quantified, in total, more than 9000 lysines in human cell proteomes and identified several hundred residues with heightened reactivity that are enriched at protein functional sites and can frequently be targeted by electrophilic small molecules. We discovered lysine-reactive fragment electrophiles that inhibit enzymes by active site and allosteric mechanisms, as well as disrupt protein-protein interactions in transcriptional regulatory complexes, emphasizing the broad potential and diverse functional consequences of liganding lysine residues throughout the human proteome.

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Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics.In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors.D escribed here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflowa nd allowed an increased coverage of cysteines in bacterial systems.They were used to quantify 59 %o fa ll cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity,w hichc orrelates with functional importance.F urthermore,2 68 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG-CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through an ew target. Overall, ab road map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modesof-action.

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Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, which do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic-acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids.