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Discovery of O-GlcNAc-6-phosphate Modified Proteins in Large-scale Phosphoproteomics Data*

  • Hannes Hahne
    Affiliations
    Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany
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  • Bernhard Kuster
    Correspondence
    To whom correspondence should be addressed: Department for Biosciences, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany. Tel.: +49 8161 715696; Fax: +49 8161 715931
    Affiliations
    Chair for Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany

    Center for Integrated Protein Science Munich, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany
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  • Author Footnotes
    * We further gratefully acknowledge the Studienstiftung des Deutschen Volkes e. V. for a PhD fellowship to HH, and the support of the Faculty Graduate Center Weihenstephan of TUM Graduate School at the Technische Universität München, Germany.
    This article contains supplemental Tables S1 and S2.
Open AccessPublished:July 23, 2012DOI:https://doi.org/10.1074/mcp.M112.019760
      Phosphorylated O-GlcNAc is a novel post-translational modification that has so far only been found on the neuronal protein AP180 from the rat (Graham et al., J. Proteome Res. 2011, 10, 2725–2733). Upon collision induced dissociation, the modification generates a highly mass deficient fragment ion (m/z 284.0530) that can be used as a reporter for the identification of phosphorylated O-GlcNAc. Using a publically available mouse brain phosphoproteome data set, we employed our recently developed Oscore software to re-evaluate high resolution/high accuracy tandem mass spectra and discovered the modification on 23 peptides corresponding to 11 mouse proteins. The systematic analysis of 220 candidate phosphoGlcNAc tandem mass spectra as well as a synthetic standard enabled the dissection of the major phosphoGlcNAc fragmentation pathways, suggesting that the modification is O-GlcNAc-6-phosphate. We find that the classical O-GlcNAc modification often exists on the same peptides indicating that O-GlcNAc-6-phosphate may biosynthetically arise in two steps involving the O-GlcNAc transferase and a currently unknown kinase. Many of the identified proteins are involved in synaptic transmission and for Ca2+/calmodulin kinase IV, the O-GlcNAc-6-phosphate modification was found in the vicinity of two autophosphorylation sites required for full activation of the kinase suggesting a potential regulatory role for O-GlcNAc-6-phosphate. By re-analyzing mass spectrometric data from human embryonic and induced pluripotent stem cells, our study also identified Zinc finger protein 462 (ZNF462) as the first human O-GlcNAc-6-phosphate modified protein. Collectively, the data suggests that O-GlcNAc-6-phosphate is a general post-translation modification of mammalian proteins with a variety of possible cellular functions.
      The attachment of N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is a dynamic post-translational modification with emerging roles in important cellular processes such as transcription, translation, cytokinesis, and signaling (
      • Hart G.W.
      • Housley M.P.
      • Slawson C.
      Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins.
      ,
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ,

      Hu, P., Shimoji, S., Hart, G. W., Site-specific interplay between O-GlcNAcylation and phosphorylation in cellular regulation. FEBS Lett. 584, 2526–2538

      ,
      • Hanover J.A.
      Epigenetics gets sweeter: O-GlcNAc joins the “histone code”.
      ). O-GlcNAcylation has been linked to phosphorylation as both modifications can occupy the same or adjacent sites (
      • Hart G.W.
      • Slawson C.
      • Ramirez-Correa G.
      • Lagerlof O.
      Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.
      ) and a functional relationship of both modifications has been identified in some cases. For instance, the interplay between O-GlcNAcylation and phosphorylation modulates the stability and activity of p53 (
      • Yang W.H.
      • Kim J.E.
      • Nam H.W.
      • Ju J.W.
      • Kim H.S.
      • Kim Y.S.
      • Cho J.W.
      Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability.
      ). However, recent data revealed the frequent co-occurrence of O-GlcNAc and phosphate at proximal sites (
      • Hahne H.
      • Gholami A.M.
      • Kuster B.
      Discovery of O-GlcNAc-modified proteins in published large-scale proteome data.
      ), suggesting the reciprocal regulation by O-GlcNAcylation and phosphorylation may not be a very general mechanism. Moreover, it has also been found that the distribution of O-GlcNAc sites relative to phosphorylation sites is rather random and that the modification rates at sites detected with both modifications are almost equal, indicating that, on a global level, the substrate recognition of both pathways is not interconnected (
      • Trinidad J.C.
      • Barkan D.T.
      • Gulledge B.F.
      • Thalhammer A.
      • Sali A.
      • Schoepfer R.
      • Burlingame A.L.
      Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.
      ).
      The identification of O-GlcNAc-modified proteins is typically achieved by combining selective enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS). In mass spectrometry based proteomics, peptides are usually analyzed by some form of collision-induced dissociation (CID). But, owing to the lability of the O-glycosidic bond under typical CID conditions, the direct and simultaneous identification of O-GlcNAc peptides and sites is difficult. Fragment ion spectra of O-GlcNAc peptides are dominated by the sugar fragments and the GlcNAc oxonium ion cannot be distinguished from other isobaric HexNAc epimers (e.g. GalNAc). Still, the fragment ions generated by the cleavage of the O-glycosidic bond define a highly useful pattern, which significantly facilitates the (automated) discovery of glycopeptides in general and O-GlcNAc peptides in particular even in complex samples (
      • Chalkley R.J.
      • Burlingame A.L.
      Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry.
      ,
      • Haynes P.A.
      • Aebersold R.
      Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography-tandem mass spectrometry.
      ,
      • Chalkley R.J.
      • Burlingame A.L.
      Identification of novel sites of O-N-acetylglucosamine modification of serum response factor using quadrupole time-of-flight mass spectrometry.
      ,
      • Vosseller K.
      • Trinidad J.C.
      • Chalkley R.J.
      • Specht C.G.
      • Thalhammer A.
      • Lynn A.J.
      • Snedecor J.O.
      • Guan S.
      • Medzihradszky K.F.
      • Maltby D.A.
      • Schoepfer R.
      • Burlingame A.L.
      O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.
      ,
      • Ozohanics O.
      • Krenyacz J.
      • Ludányi K.
      • Pollreisz F.
      • Vćkey K.
      • Drahos L.
      GlycoMiner: a new software tool to elucidate glycopeptide composition.
      ,
      • Pompach P.
      • Chandler K.B.
      • Lan R.
      • Edwards N.
      • Goldman R.
      Semi-automated identification of N-Glycopeptides by hydrophilic interaction chromatography, nano-reverse-phase LC-MS/MS, and glycan database search.
      ,
      • Zhao P.
      • Viner R.
      • Teo C.F.
      • Boons G.J.
      • Horn D.
      • Wells L.
      Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.
      ). The specificity of these diagnostic fragment ions is further increased when identified from high resolution and high mass accuracy tandem MS spectra (
      • Zhao P.
      • Viner R.
      • Teo C.F.
      • Boons G.J.
      • Horn D.
      • Wells L.
      Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.
      ,
      • Hahne H.
      • Kuster B.
      A novel two-stage tandem mass spectrometry approach and scoring scheme for the identification of O-GlcNAc modified peptides.
      ). To interrogate such data systematically, we have recently developed a simple scoring scheme, termed Oscore, which automatically assesses tandem mass spectra for the presence and intensity of O-GlcNAc (HexNAc) diagnostic fragment ions and, in turn, allows ranking spectra according their probability of representing an O-GlcNAc peptide (
      • Hahne H.
      • Kuster B.
      A novel two-stage tandem mass spectrometry approach and scoring scheme for the identification of O-GlcNAc modified peptides.
      ). A combined search strategy using the protein identification software Mascot and our Oscore algorithm enabled the identification of hundreds of O-GlcNAc peptides from large-scale proteome data (
      • Hahne H.
      • Gholami A.M.
      • Kuster B.
      Discovery of O-GlcNAc-modified proteins in published large-scale proteome data.
      ).
      Very recently, phosphorylated O-GlcNAc (phosphoGlcNAc) has been identified for the first time on the synapse-specific protein AP180 purified from rat brain (
      • Graham M.E.
      • Thaysen-Andersen M.
      • Bache N.
      • Craft G.E.
      • Larsen M.R.
      • Packer N.H.
      • Robinson P.J.
      A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation.
      ). In light of this exciting discovery, we here report on the extension of the combined Mascot/Oscore approach for the discovery of proteins modified with phosphoGlcNAc. We first adapted the Oscore for the detection of phosphoGlcNAc and then re-assessed a large-scale phosphoproteomic data set from murine brain (
      • Jedrychowski M.P.
      • Huttlin E.L.
      • Haas W.
      • Sowa M.E.
      • Rad R.
      • Gygi S.P.
      Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics.
      ). This led to the discovery of 23 phosphoGlcNAc peptides on 11 phosphoGlcNAc proteins. Based on the fragmentation patterns of 220 candidate phosphoGlcNAc spectra and a synthetic standard, we deduced O-GlcNAc-6-phosphate as the most likely molecular entity. Finally, the re-analysis of a phosphoproteome study of human embryonic (hES) and induced pluripotent stem (iPS) cells (
      • Phanstiel D.H.
      • Brumbaugh J.
      • Wenger C.D.
      • Tian S.
      • Probasco M.D.
      • Bailey D.J.
      • Swaney D.L.
      • Tervo M.A.
      • Bolin J.M.
      • Ruotti V.
      • Stewart R.
      • Thomson J.A.
      • Coon J.J.
      Proteomic and phosphoproteomic comparison of human ES and iPS cells.
      ), revealed evidence for the first time that a human protein may be modified by O-GlcNAc-6-phosphate suggesting that this PTM may exist more generally in mammalian systems.

      CONCLUSIONS

      The Oscore-based re-assessment of high resolution tandem mass spectra from published phosphoproteomic studies enabled the identification of 12 O-GlcNAc-6-phosphate modified proteins, including the first human O-GlcNAc-6-phosphate modified protein. This shows that O-GlcNAc-6-phosphate is not a singular protein modification (
      • Graham M.E.
      • Thaysen-Andersen M.
      • Bache N.
      • Craft G.E.
      • Larsen M.R.
      • Packer N.H.
      • Robinson P.J.
      A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation.
      ) and that it is sufficiently stable and abundant to be detected in the presence of tens of thousands of phosphopeptides. Thus we expect that mining phosphoprotemic data will substantially increase the number of proteins that can be modified in this way. Still, more efficient biochemical enrichment tools as well as MS techniques such as ETD that preserves the modification will likely be required for the proteome-wide investigation of O-GlcNAc-6-phosphate in the future. In addition to merely enumerating modified peptides, the identification of the corresponding O-GlcNAc kinase(s) as well as potentially involved phosphatases will clearly be important steps toward a basic understanding of this novel post-translational modification.

      Acknowledgments

      We thank the originators of the mass spectrometry data used in this study for making their data available to the community.

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