Investigating Peptide‐Coenzyme A Conjugates as Chemical Probes for Proteomic Profiling of N‐Terminal and Lysine Acetyltransferases

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. The α‐Ala‐CoA probe enriched the majority of NAT catalytic and auxiliary subunits, while a lysine CoA‐conjugate 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 and 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.