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 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.