Abstract
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.
Highlights
Novel gel, proteomic and ELISA-based methods to determine N-myristoylation of proteins in cells, without metabolic labelling
YnMyr tagging has superior identification power for NMT substrates thanks to selective enrichment of myristoylated proteins, our Sortase A (SrtA) approach successfully detects the response to NMT inhibition for most (81%) of the NMT substrates identified by both methods
We hypothesize that where SrtA does not detect a response to inhibition this is most likely because of the requirement for an exposed N-terminal glycine which can be affected by changes in structural conformation, or N-terminal modifications including proteolysis or N-terminal acetylation (30, 31), the latter is relatively rare at N-terminal Gly in human cells (32)
Summary
Novel gel, proteomic and ELISA-based methods to determine N-myristoylation of proteins in cells, without metabolic labelling. We and others have applied chemically tagged myristate analogues that are cell-permeable and readily transferred by endogenous NMT onto protein substrates to allow for visual assessment of NMT activity or enrichment of tagged proteins upon chemical ligation to fluorophores or affinity reagents in vitro (8). This method has proven to be very powerful for substrate identification and NMT activity profiling in cells (8, 9), it relies on metabolic tagging with a chemically modified (e.g. alkynylated) analogue.
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