Abstract

Benzoylation (Kbz) is a physiologically relevant post-translational modification derived from the food additive sodium benzoate. While Kbz has been implicated in unique cellular regulatory processes, its substrate landscape and functional consequences remain poorly characterized. Conventional antibody-based enrichment methods for Kbz detection suffer from affinity bias and limited specificity. Here, we developed AyBz3, a bioorthogonal chemical probe enabling the unbiased mapping of Kbz across the proteome. Implementation of AyBz3 in HepG2 cells revealed 688 unique Kbz sites, significantly expanding the known benzoylome. Functional analysis revealed that Kbz-modified proteins are enriched in pathways related to protein translation and cell adhesion. Notably, we demonstrated that Kbz modification of nucleophosmin 1 (NPM1) impairs its molecular chaperone function toward p53, resulting in accelerated p53 degradation. Together, this study establishes AyBz3 as a powerful probe for unbiased benzoylome profiling and provides new insights into the regulatory roles of Kbz in cellular processes.