Updated PubMed. Acetylation of amino groups is a prevalent protein modification in all kingdoms of life. Acetyl groups are transferred from Coenzyme A (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 N-terminal CoA-conjugated peptide probes for interactome profiling of NAT complexes. These probes specifically recruited catalytic and auxiliary subunits of the major NAT complexes from cell lysates. When comparing the specificity of N-terminal and lysine CoA-conjugated probes the latter bound only a subset of endogenous KATs and appeared more prone to recruitment of other CoA-binding proteins. Western blot validation confirmed the specificity of selected NATs and KATs towards these probes supporting the notion that N-terminal CoA-conjugated peptides are versatile probes for NAT complex profiling in lysates of physiological and pathological backgrounds.