[¹⁴C]-Acetyl-Coenzyme A-Based In Vitro N-Terminal Acetylation Assay

N-terminal acetylation is one of the most abundant co- and posttranslational protein modifications, conserved from prokaryotes to eukaryotes. The functional consequences of this modification are manifold, ranging from protein folding, stability, and interaction to subcellular localization. We describe here an isotope-labeled [¹⁴C]-acetyl-Coenzyme A-based acetylation assay, allowing the determination of weak catalytic activities of NATs in vitro. It allows the use of purified recombinant enzymes from Escherichia coli, or co-immunoprecipitated enzymes from various organisms, as well as the determination of the in vitro activity of various cell lysates. Although marked as an old-fashioned biochemical approach, it is the ideal method to hunt for catalytic activities and defining peptide specificities of new potential N-terminal acetyltransferase candidates.