Whole Proteome Profiling of N-Myristoyltransferase Activity and Inhibition Using Sortase A

Mol Cell Proteomics. 2019 Jan;18(1):115-126. doi: 10.1074/mcp.RA118.001043. Epub 2018 Oct 19.


N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging.

Keywords: Chemical Biology; Drug Targets; IMP-1088; N-Myristoylation; N-terminal Modifications; NMT Inhibitor; Sortase A; Substrate Identification; Tandem Mass Spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / metabolism*
  • Aminoacyltransferases / metabolism*
  • Bacterial Proteins / metabolism*
  • Cell Line, Tumor
  • Chromatography, Liquid
  • Cysteine Endopeptidases / metabolism*
  • Glycine / metabolism*
  • HeLa Cells
  • Humans
  • Proteomics / methods*
  • Staphylococcus aureus / enzymology*
  • Substrate Specificity
  • Tandem Mass Spectrometry


  • Bacterial Proteins
  • Acyltransferases
  • glycylpeptide N-tetradecanoyltransferase
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases
  • Glycine