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

A new method to quantify cellular myristoylation at physiological levels and in response to N-myristoyltransferase inhibition is presented and validated. Sortase A is an effective tool to label protein glycine N-termini across the whole proteome, and its specificity is determined in this context. Generally applicable improvements to the biotin/avidin affinity enrichment protocol are described that effectively eliminate avidin-derived tryptic peptide contaminants. [Display omitted] Highlights •Application of Sortase A to label protein N-termini across the whole proteome.•Novel gel, proteomic and ELISA-based methods to determine N-myristoylation of proteins in cells, without metabolic labelling.•Side by side mass spectrometric quantification of changes in protein N-myristoylation by two complementary methods.•Improved Biotin-Neutravidin affinity enrichment protocol. 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, provi