Profiling the proteome-wide selectivity of diverse electrophiles

Covalent inhibitors that do not rely on hijacking enzymatic activity have mainly been limited to those targeting cysteine residues. The development of such cysteine-directed covalent inhibitors has greatly profited from the use of competitive residue-specific proteomics to determine their proteome-wide selectivity. Several probes have been developed to monitor other amino acids using this technology, and many more electrophiles exist to modify proteins. Nevertheless, there has been a lack of direct, proteome-wide comparisons of the selectivity of diverse electrophiles. Here we developed an unbiased workflow to analyse electrophile selectivity proteome-wide and used it to directly compare 56 alkyne probes containing diverse reactive groups. In this way, we verified and identified probes to monitor a total of nine different amino acids, as well as the protein amino terminus, across the proteome.