Updated PubMed. Acetyl groups are transferred from Acetyl-Coenzyme A (Ac-CoA) to protein N-termini and lysine side chains by N-terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs), respectively. Building on lysine-CoA conjugates as KAT probes, we have synthesized peptide probes with CoA conjugated to N-terminal alanine (α-Ala-CoA), proline (α-Pro-CoA) or tri-glutamic acid (α-3Glu-CoA) units for interactome profiling of NAT complexes. These probes recruited catalytic and auxiliary subunits of the major NAT complexes from cell lysates specifically. The α-Ala-CoA probe was more specific in recruiting NAT catalytic and auxiliary subunits when compared to a lysine CoA-conjugate which bound only a subset of endogenous KATs. Interactome profiling with the α-Pro-CoA probe showed reduced NAT recruitment in favor of metabolic CoA binding proteins, while α-3Glu-CoA steered the interactome towards NAA80 and NatB. These findings agreed with the inherent substrate specificities of the target proteins and showed that N-terminal CoA-conjugated peptides are versatile probes for NAT complex profiling in lysates of physiological and pathological backgrounds.