Chemical Proteomic Profiling of Lysine Benzoylation

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 <b>AyBz3</b>, a bioorthogonal chemical probe enabling the unbiased mapping of Kbz across the proteome. Implementation of <b>AyBz3</b> 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 <b>AyBz3</b> as a powerful probe for unbiased benzoylome profiling and provides new insights into the regulatory roles of Kbz in cellular processes.