Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

Proc Natl Acad Sci U S A. 2012 May 8;109(19):7280-5. doi: 10.1073/pnas.1200425109. Epub 2012 Apr 19.


O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetylglucosamine / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Brain / enzymology*
  • Brain / metabolism
  • Cell Membrane / metabolism
  • Cell Nucleus / metabolism
  • Cytoplasm / metabolism
  • Epidermal Growth Factor / metabolism
  • Glycoproteins / metabolism
  • Glycosylation
  • Mice
  • Molecular Sequence Data
  • N-Acetylglucosaminyltransferases / metabolism*
  • Organelles / metabolism
  • Peptides / metabolism
  • Phosphorylation
  • Proteome / metabolism
  • Proteomics / methods
  • Tandem Mass Spectrometry / methods*


  • Glycoproteins
  • Peptides
  • Proteome
  • Epidermal Growth Factor
  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • Acetylglucosamine