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Whole proteome profiling of N-myristoyltransferase activity and inhibition using Sortase A

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Title: Whole proteome profiling of N-myristoyltransferase activity and inhibition using Sortase A
Authors: Goya Grocin, A
Serwa, R
Morales Sanfrutos, J
Ritzefeld, M
Tate, E
Item Type: Journal Article
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.
Issue Date: 2-Jan-2019
Date of Acceptance: 19-Oct-2018
URI: http://hdl.handle.net/10044/1/65607
DOI: https://dx.doi.org/10.1074/mcp.RA118.001043
ISSN: 1535-9476
Publisher: American Society for Biochemistry and Molecular Biology
Start Page: 115
End Page: 126
Journal / Book Title: Molecular and Cellular Proteomics
Volume: 18
Issue: 1
Copyright Statement: © 2019 Grocin et al. Published by the American Society for Biochemistry and Molecular Biology, Inc.
Keywords: Chemical biology
Drug targets*
N-terminal modifications*
NMT inhibitor
Sortase A
Substrate identification
Tandem Mass Spectrometry
MD Multidisciplinary
Biochemistry & Molecular Biology
Publication Status: Published
Online Publication Date: 2018-10-19
Appears in Collections:Chemistry
Biological and Biophysical Chemistry
Faculty of Natural Sciences