Fig. 6: MS-based approaches for the identification of NAT substrates and substrate specificities.

a The main steps of the workflow for SILProNAQ and COFRADIC. A protein mixture is trideutero-acetylated on free amine groups (N-termini or lysine residues (K)), digested with trypsin, and solid cation exchange (SCX) fractionated to remove free N-termini. In COFRADIC, a two-step fractionation scheme is performed at this stage (not shown). Peptides are then quantified by LC/MS, and the magnitude of intensity change in light/heavy Nt-acetyl groups (non-deuterated vs. trideuterated Nt-acetyl) measures the fraction of acetylation for a given N-terminus. b The generation of peptides for an in vitro peptide library is performed in the same manner as that of COFRADIC/SILProNAQ, except that at the SCX stage, peptides with free N-termini are retained rather than being discarded and are used as substrates for recombinant NAT enzymes. The output of this reaction is measured by LC/MS and is used to deduce the substrate preference of the NAT in question