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 (alpha-Ala-CoA), proline (alpha-Pro-CoA) or tri-glutamic acid (alpha-3Glu-CoA) units for interactome profiling of NAT complexes. The alpha-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 alpha-Pro-CoA probe showed reduced NAT recruitment in favor of metabolic CoA binding proteins and alpha-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.