A fluorescent CPM-based in vitro acetylation assay: A tool for assessing N-terminal acetyltransferase activity and...

N-terminal acetylation is a prevalent co-translational modification in eukaryotes, affecting ∼80 % of human cytosolic proteins. Catalyzed by N-terminal acetyltransferases (NATs), this irreversible modification regulates protein stability, localization, and interactions. Despite its importance, high-throughput, non-radioactive methods to study NAT activity remain limited. We present an optimized fluorescence-based assay using 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to monitor NAT activity via real-time detection of free coenzyme A released during acetyl transfer. We established a workflow for human NatA and NatB, including enzyme purification, reaction setup, and assay readout. The assay shows high sensitivity and a linear correlation between CPM fluorescence and coenzyme A, enabling accurate kinetic measurements and inhibitor testing in a 96-well format. It was miniaturized to 384- and 1536-well formats and validated using a bisubstrate inhibitor. Optimization steps for enzyme and substrate concentrations and time-course analysis are provided. Assay interferences are discussed, and orthogonal validation strategies are recommended to confirm inhibitor specificity in follow-up applications.