N-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes and is catalysed in humans by seven N-acetyltransferases. AtNAA60 localizes to the plasma membrane in vivo by an α-helical membrane anchor at its C-terminus. In this study, we investigated the Arabidopsis thaliana N-terminal acetylome in leaf cells of naa60-1 to identify AtNAA60 substrates in vivo using the SILProNAQ approach.