Discovery of O-GlcNAc-6-phosphate modified proteins in large-scale phosphoproteomics data

Mol Cell Proteomics. 2012 Oct;11(10):1063-9. doi: 10.1074/mcp.M112.019760. Epub 2012 Jul 23.


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 Ca(2+)/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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Amino Acid Sequence
  • Animals
  • Brain / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinase Type 4 / genetics
  • Calcium-Calmodulin-Dependent Protein Kinase Type 4 / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Humans
  • Mice
  • Molecular Sequence Data
  • N-Acetylglucosaminyltransferases / metabolism
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Peptides / analysis
  • Phosphorylation
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism
  • Protein Processing, Post-Translational*
  • Rats
  • Software*
  • Synaptic Transmission / genetics
  • Tandem Mass Spectrometry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Zinc Fingers / genetics


  • DNA-Binding Proteins
  • Nerve Tissue Proteins
  • Peptides
  • Transcription Factors
  • ZNF462 protein, human
  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • Calcium-Calmodulin-Dependent Protein Kinase Type 4
  • Acetylglucosamine