Whole proteome profiling of N-Myristoyltransferase activity and inhibition using sortase A

Mostra el registre complet Registre parcial de l'ítem

• dc.contributor.author Goya Grocin, Andrea
• dc.contributor.author Serwa, Remigiusz A.
• dc.contributor.author Morales Sanfrutos, Julia
• dc.contributor.author Ritzefeld, Markus
• dc.contributor.author Tate, Edward W.
• dc.date.accessioned 2020-04-09T07:50:14Z
• dc.date.available 2020-04-09T07:50:14Z
• dc.date.issued 2019
• dc.description.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.
• dc.format.mimetype application/pdf
• dc.identifier.citation Goya Grocin A, Serwa RA, Morales Sanfrutos J, Ritzefeld M, Tate EW. Whole proteome profiling of N-Myristoyltransferase activity and inhibition using sortase A. Mol Cell Proteomics. 2019; 18(1):115-26. DOI: 10.1074/mcp.RA118.001043
• dc.identifier.doi http://dx.doi.org/10.1074/mcp.RA118.001043
• dc.identifier.issn 1535-9476
• dc.identifier.uri http://hdl.handle.net/10230/44193
• dc.language.iso eng
• dc.publisher American Society for Biochemistry and Molecular Biology (ASBMB)
• dc.relation.ispartof Mol Cell Proteomics. 2019; 18(1):115-26
• dc.rights © 2019 Grocin et al. Published by the American Society for Biochemistry and MolecularBiology, Inc. Final version open access under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0)
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0
• dc.subject.keyword Chemical biology
• dc.subject.keyword Drug targets
• dc.subject.keyword IMP-1088
• dc.subject.keyword N-Myristoylation
• dc.subject.keyword N-terminal modifications
• dc.subject.keyword NMT inhibitor
• dc.subject.keyword Sortase A
• dc.subject.keyword Substrate identification
• dc.subject.keyword Tandem mass spectrometry
• dc.title Whole proteome profiling of N-Myristoyltransferase activity and inhibition using sortase A
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion