HPLC-based quantification of in vitro N-terminal acetylation.

Protein N-terminal acetylation is a widespread modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs). The various NATs and their specific substrate specificities and catalytic mechanisms are far from fully understood. We here describe an in vitro method based on reverse-phase HPLC to quantitatively measure in vitro acetylation of NAT oligopeptide substrates, enabling the determination of NAT specificity as well as kinetic parameters.