Detecting the "O-GlcNAc-ome"; detection, purification, and analysis of O-GlcNAc modified proteins

Methods Mol Biol. 2009;534:251-79. doi: 10.1007/978-1-59745-022-5_19.


The modification of Ser and Thr residues of cytoplasmic and nuclear proteins with a monosaccharide of O-linked beta-N-acetylglucosamine is an essential and dynamic post-translational modification of metazoans. Deletion of the O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, is lethal in mammalian cells highlighting the importance of this post-translational modification in regulating cellular function. O-GlcNAc is believed to modulate protein function in a manner analogous to protein phosphorylation. Notably, on some proteins O-GlcNAc and O-phosphate modify the same Ser/Thr residue, suggesting that a reciprocal relationship exists between these two post-translational modifications. In this chapter we describe the most robust techniques for the detection and purification of O-GlcNAc modified proteins, and discuss some more specialized techniques for site-mapping and detection of O-GlcNAc during mass spectrometry.

Publication types

  • Research Support, N.I.H., Extramural
  • Review

MeSH terms

  • Acetylglucosamine / analysis*
  • Acetylglucosamine / isolation & purification*
  • Acetylglucosamine / metabolism
  • Animals
  • Cell Culture Techniques
  • Glycomics / methods*
  • Glycoproteins / analysis*
  • Glycoproteins / metabolism
  • Humans
  • Immunoblotting / methods
  • Models, Biological
  • N-Acetylglucosaminyltransferases / metabolism
  • Staining and Labeling / methods


  • Glycoproteins
  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • Acetylglucosamine