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

Abstract

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 Nterminal 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.

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“…We acquired the mass spectra in data-dependent acquisition mode as outlined in Sindlinger et al . [ 19 ]. We processed the raw data, by using the MaxQuant software version 2.0.3.0 [ 20 ].…”

“…We separated peptides on 20 cm frit-less silica emitters (CoAnn Technologies, 0.75 µm inner diameter), packed inhouse with reversed-phase ReproSil-Pur C 18 AQ 1.9 µm resin (Dr Maisch) and kept the column constantly at 50°C. We acquired the mass spectra in data-dependent acquisition mode as outlined in Sindlinger et al [19]. We processed the raw data, by using the MaxQuant software version 2.0.3.0 [20].…”