Purification and activity assays of N-terminal acetyltransferase D

α-N-terminal acetyltransferase D (NatD) specifically acetylates the α-amine of the N-terminal SGRGK motif on histones H4 and H2A, a modification crucial for regulating gene expression. Thus, assays for the characterization of NatD-mediated acetylation is essential to study its functions and discover inhibitors. Here we present two methods for characterizing NatD-mediated acetylation: fluorescence-based and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) assays. We describe detailed procedures to characterize enzymatic kinetics and assess inhibitor activity for NatD. Additionally, we discuss the advantages and limitations of each approach compared to other in vitro acetyltransferase assays and highlight their broader applicability for evaluating N-terminal acetylation.