Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry

Hannes Hahne, Nadine Sobotzki, Tamara Nyberg, Dominic Helm, Vladimir S. Borodkin, Daan M. F. van Aalten, Brian Agnew, Bernhard Kuster

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    93 Citations (Scopus)

    Abstract

    The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective beta-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.

    Original languageEnglish
    Pages (from-to)927-936
    Number of pages10
    JournalJournal of Proteome Research
    Volume12
    Issue number2
    DOIs
    Publication statusPublished - 2013

    Keywords

    • ENERGY SENSOR
    • PHOSPHORYLATION
    • GLOBAL IDENTIFICATION
    • LINKED N-ACETYLGLUCOSAMINE
    • ELECTRON-TRANSFER DISSOCIATION
    • POSTTRANSLATIONAL MODIFICATIONS
    • PEPTIDE IDENTIFICATION
    • metabolic labeling
    • GLCNACYLATION
    • GLYCOSYLATION
    • mass spectrometry
    • post-translational modifications
    • O-GlcNAc
    • proteomics
    • BETA-ELIMINATION
    • Click chemistry
    • beta-elimination

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