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Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag

Griffin, Matthew E. and Jensen, Elizabeth H. and Mason, Daniel E. and Jenkins, Courtney L. and Stone, Shannon E. and Peters, Eric C. and Hsieh-Wilson, Linda C. (2016) Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag. Molecular BioSystems, 2016 (6). pp. 1756-1759. ISSN 1742-206X. PMCID PMC4905554. http://resolver.caltech.edu/CaltechAUTHORS:20160413-083438795

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Abstract

The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(I)-catalysed azide–alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows subsequent quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT), as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1039/C6MB00138FDOIArticle
http://pubs.rsc.org/en/Content/ArticleLanding/2016/MB/C6MB00138FPublisherArticle
http://www.rsc.org/suppdata/c6/mb/c6mb00138f/c6mb00138f1.pdfPublisherSupplementary Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905554/PubMed CentralArticle
ORCID:
AuthorORCID
Stone, Shannon E.0000-0002-6617-3874
Hsieh-Wilson, Linda C.0000-0001-5661-1714
Additional Information:© 2016 Royal Society of Chemistry. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Received 22nd February 2016, Accepted 25th March 2016. First published online 30 Mar 2016. This article is part of themed collection: 2016 Hot Articles in Molecular BioSystems and Protein Labelling. We thank Dr. M. Shahgholi for assistance with peptide analysis by LC-MS. This research was supported by the National Institutes of Health (R01-GM084724), the National Science Foundation (GRFP DGE-1144469, M. E. G.), and the Department of Defense (NDSEG, E. H. J.).
Funders:
Funding AgencyGrant Number
NIHR01-GM084724
NSF Graduate Research FellowshipDGE-1144469
National Defense Science and Engineering Graduate (NDSEG) FellowshipUNSPECIFIED
PubMed Central ID:PMC4905554
Record Number:CaltechAUTHORS:20160413-083438795
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160413-083438795
Official Citation:Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag Matthew E. Griffin, Elizabeth H. Jensen, Daniel E. Mason, Courtney L. Jenkins, Shannon E. Stone, Eric C. Peters and Linda C. Hsieh-Wilson Mol. BioSyst., 2016,12, 1756-1759 DOI: 10.1039/C6MB00138F
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66100
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:13 Apr 2016 16:37
Last Modified:06 Nov 2017 21:35

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