Bromo-desthiobiotin (Br-DTB) tag for unbiased profiling and identification of chemical modifications

Leveraging the intrinsic reactivity of cysteine, isoTOP-ABPP (activity-based protein profiling) has been a powerful chemoproteomic platform for cysteine quantification, ligand, and chemical modification discovery. Here, we report new bromo-desthiobiotin azide (Br-DTB) tags for covalent labelling, enabling unbiased identification of chemical modifications. Besides peptide identification from MS2 spectra, Br's isotopic MS1 signatures provide an additional layer of validation to confirm labelled sites and eliminate false-positive assignments. To support this analysis, we have developed a publicly available in-house software to automatically recognize this isotopic signature. The accuracy of the Br-DTB tag was further validated by a synthetic peptide in vitro and l-azidohomoalanine (AHA) labelling in living cells. We further elucidated the covalent labelling site of piperlongumine (PL) on the E3 ligase KEAP1, demonstrating its utility for discovering new covalent labelling sites. Collectively, Br-DTB tags are suitable for chemoproteomic workflows, providing a robust platform for covalent labelling discovery and functional proteome profiling.