Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics


The addition of the monosaccharide β-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Accurate quantification of known O-GlcNAc peptides from complex mixtures using the QUIC-Tag approach.
Figure 2: O-GlcNAc glycosylation is reversible in cultured cortical neurons.
Figure 3: Sequencing of tagged O-GlcNAc peptides regulated by PUGNAc treatment using CAD.
Figure 4: Sequencing of tagged O-GlcNAc peptides regulated by PUGNAc treatment using ETD.
Figure 5: Quantification of O-GlcNAc glycosylation on intact proteins by immunoblotting and infrared imaging detection.
Figure 6: O-GlcNAc glycosylation is dynamically modulated by robust excitatory stimulation of the brain in vivo using kainic acid.


  1. 1

    Khidekel, N. & Hsieh-Wilson, L.C. A 'molecular switchboard'–covalent modifications to proteins and their impact on transcription. Org. Biomol. Chem. 2, 1–7 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Greengard, P. The neurobiology of slow synaptic transmission. Science 294, 1024–1030 (2001).

    CAS  Article  Google Scholar 

  3. 3

    Love, D.C. & Hanover, J.A. The hexosamine signaling pathway: deciphering the “O-GlcNAc code”. Sci. STKE 2005, re13 (2005).

    Google Scholar 

  4. 4

    Khidekel, N., Ficarro, S.B., Peters, E.C. & Hsieh-Wilson, L.C. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc. Natl. Acad. Sci. USA 101, 13132–13137 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Vosseller, K. et al. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 5, 923–934 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Kneass, Z.T. & Marchase, R.B. Neutrophils exhibit rapid agonist-induced increases in protein-associated O-GlcNAc. J. Biol. Chem. 279, 45759–45765 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Zachara, N.E. et al. Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J. Biol. Chem. 279, 30133–30142 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Cheng, X. & Hart, G.W. Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor beta: post-translational regulation of turnover and transactivation activity. J. Biol. Chem. 276, 10570–10575 (2001).

    CAS  Article  Google Scholar 

  9. 9

    Chou, T.Y., Hart, G.W. & Dang, C.V. c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas. J. Biol. Chem. 270, 18961–18965 (1995).

    CAS  Article  Google Scholar 

  10. 10

    Iyer, S.P. & Hart, G.W. Dynamic nuclear and cytoplasmic glycosylation: enzymes of O-GlcNAc cycling. Biochemistry 42, 2493–2499 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Cole, R.N. & Hart, G.W. Cytosolic O-glycosylation is abundant in nerve terminals. J. Neurochem. 79, 1080–1089 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Iyer, S.P.N. & Hart, G.W. Dynamic nuclear and cytoplasmic glycosylation: enzymes of O- GlcNAc cycling. Biochemistry 42, 2493–2499 (2003).

    CAS  Article  Google Scholar 

  13. 13

    O'Donnell, N., Zachara, N.E., Hart, G.W. & Marth, J.D. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Mol. Cell. Biol. 24, 1680–1690 (2004).

    CAS  Article  Google Scholar 

  14. 14

    Lamarre-Vincent, N. & Hsieh-Wilson, L.C. Dynamic glycosylation of the transcription factor CREB: a potential role in gene regulation. J. Am. Chem. Soc. 125, 6612–6613 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Griffith, L.S. & Schmitz, B. O-linked N-acetylglucosamine levels in cerebellar neurons respond reciprocally to perturbations of phosphorylation. Eur. J. Biochem. 262, 824–831 (1999).

    CAS  Article  Google Scholar 

  16. 16

    Khidekel, N. et al. A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162–16163 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Ong, S.E., Mittler, G. & Mann, M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat. Methods 1, 119–126 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Ross, P.L. et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154–1169 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Tai, H.C., Khidekel, N., Ficarro, S.B., Peters, E.C. & Hsieh-Wilson, L.C. Parallel identification of O-GlcNAc-modified proteins from cell lysates. J. Am. Chem. Soc. 126, 10500–10501 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Cieniewski-Bernard, C. et al. Identification of O-linked N-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry. Mol. Cell. Proteomics 3, 577–585 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Hsu, J.L., Huang, S.Y., Chow, N.H. & Chen, S.H. Stable-isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843–6852 (2003).

    CAS  Article  Google Scholar 

  22. 22

    Chalkley, R.J. & Burlingame, A.L. Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 1106–1113 (2001).

    CAS  Article  Google Scholar 

  23. 23

    Makarov, A., Denisov, E., Lange, O. & Horning, S. Dynamic range of mass accuracy in LTQ orbitrap hybrid mass spectrometer. J. Am. Soc. Mass Spectrom. 17, 977–982 (2006).

    CAS  Article  Google Scholar 

  24. 24

    Roquemore, E.P., Chevrier, M.R., Cotter, R.J. & Hart, G.W. Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin. Biochemistry 35, 3578–3586 (1996).

    CAS  Article  Google Scholar 

  25. 25

    Zhang, R., Sioma, C.S., Wang, S. & Regnier, F.E. Fractionation of isotopically labeled peptides in quantitative proteomics. Anal. Chem. 73, 5142–5149 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Haltiwanger, R.S., Grove, K. & Philipsberg, G.A. Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate. J. Biol. Chem. 273, 3611–3617 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J. & Hunt, D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. USA 101, 9528–9533 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Coon, J.J., Syka, J.E.P., Schwartz, J.C., Shabanowitz, J. & Hunt, D.F. Anion dependence in the partitioning between proton and electron transfer in ion/ion reactions. Int. J. Mass Spectrom. 236, 33–42 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Hogan, J.M., Pitteri, S.J., Chrisman, P.A. & McLuckey, S.A. Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced dissociation. J. Proteome Res. 4, 628–632 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Swaney, D.L. et al. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Anal. Chem. 79, 477–485 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Lazarus, B.D., Love, D.C. & Hanover, J.A. Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates. Glycobiology 16, 415–421 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Whisenhunt, T.R. et al. Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development. Glycobiology 16, 551–563 (2006).

    CAS  Article  Google Scholar 

  33. 33

    Nedivi, E., Hevroni, D., Naot, D., Israeli, D. & Citri, Y. Numerous candidate plasticity-related genes revealed by differential cDNA cloning. Nature 363, 718–722 (1993).

    CAS  Article  Google Scholar 

  34. 34

    Ben-Ari, Y. & Cossart, R. Kainate, a double agent that generates seizures: two decades of progress. Trends Neurosci. 23, 580–587 (2000).

    CAS  Article  Google Scholar 

  35. 35

    Beckmann, A.M., Davidson, M.S., Goodenough, S. & Wilce, P.A. Differential expression of Egr-1-like DNA-binding activities in the naive rat brain and after excitatory stimulation. J. Neurochem. 69, 2227–2237 (1997).

    CAS  Article  Google Scholar 

  36. 36

    Nandi, A. et al. Global identification of O-GlcNAc-modified proteins. Anal. Chem. 78, 452–458 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Kamemura, K., Hayes, B.K., Comer, F.I. & Hart, G.W. Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitogens. J. Biol. Chem. 277, 19229–19235 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Ludemann, N. et al. O-glycosylation of the tail domain of neurofilament protein M in human neurons and in spinal cord tissue of a rat model of amyotrophic lateral sclerosis (ALS). J. Biol. Chem. 280, 31648–31658 (2005).

    Article  Google Scholar 

  39. 39

    Vosseller, K. et al. Quantitative analysis of both protein expression and serine/ threonine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5, 388–398 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Brackertz, M., Gong, Z., Leers, J. & Renkawitz, R. p66alpha and p66beta of the Mi-2/NuRD complex mediate MBD2 and histone interaction. Nucleic Acids Res. 34, 397–406 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Yang, X. et al. O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability. Proc. Natl. Acad. Sci. USA 98, 6611–6616 (2001).

    CAS  Article  Google Scholar 

  42. 42

    Gong, Z., Brackertz, M. & Renkawitz, R. SUMO modification enhances p66-mediated transcriptional repression of the Mi-2/NuRD complex. Mol. Cell. Biol. 26, 4519–4528 (2006).

    CAS  Article  Google Scholar 

  43. 43

    Ule, J. & Darnell, R.B. RNA binding proteins and the regulation of neuronal synaptic plasticity. Curr. Opin. Neurobiol. 16, 102–110 (2006).

    CAS  Article  Google Scholar 

  44. 44

    Elvira, G., Massie, B. & DesGroseillers, L. The zinc-finger protein ZFR is critical for Staufen 2 isoform specific nucleocytoplasmic shuttling in neurons. J. Neurochem. 96, 105–117 (2006).

    CAS  Article  Google Scholar 

  45. 45

    Bastos, R., Lin, A., Enarson, M. & Burke, B. Targeting and function in mRNA export of nuclear pore complex protein Nup153. J. Cell Biol. 134, 1141–1156 (1996).

    CAS  Article  Google Scholar 

  46. 46

    Jones, M.W. et al. A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories. Nat. Neurosci. 4, 289–296 (2001).

    CAS  Article  Google Scholar 

  47. 47

    Thiel, G. & Cibelli, G. Regulation of life and death by the zinc finger transcription factor Egr-1. J. Cell. Physiol. 193, 287–292 (2002).

    CAS  Article  Google Scholar 

  48. 48

    James, A.B., Conway, A.M. & Morris, B.J. Genomic profiling of the neuronal target genes of the plasticity-related transcription factor – Zif268. J. Neurochem. 95, 796–810 (2005).

    CAS  Article  Google Scholar 

  49. 49

    Wang, Q., Yu, S., Simonyi, A., Sun, G.Y. & Sun, A.Y. Kainic acid-mediated excitotoxicity as a model for neurodegeneration. Mol. Neurobiol. 31, 3–16 (2005).

    CAS  Article  Google Scholar 

  50. 50

    Marin, P. et al. Glutamate-dependent phosphorylation of elongation factor-2 and inhibition of protein synthesis in neurons. J. Neurosci. 17, 3445–3454 (1997).

    CAS  Article  Google Scholar 

  51. 51

    Datta, R., Choudhury, P., Ghosh, A. & Datta, B. A glycosylation site, 60SGTS63, of p67 is required for its ability to regulate the phosphorylation and activity of eukaryotic initiation factor 2α. Biochemistry 42, 5453–5460 (2003).

    CAS  Article  Google Scholar 

  52. 52

    Collins, M.O. et al. Proteomic analysis of in vivo phosphorylated synaptic proteins. J. Biol. Chem. 280, 5972–5982 (2005).

    CAS  Article  Google Scholar 

  53. 53

    Gu, Y., Hamajima, N. & Ihara, Y. Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser-518, and Ser-522. Biochemistry 39, 4267–4275 (2000).

    CAS  Article  Google Scholar 

  54. 54

    Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G.W. & Gong, C.X. O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease. Proc. Natl. Acad. Sci. USA 101, 10804–10809 (2004).

    CAS  Article  Google Scholar 

  55. 55

    Gama, C.I. et al. Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat. Chem. Biol. 2, 467–473 (2006).

    CAS  Article  Google Scholar 

Download references


We thank P. Qasba and B. Ramakrishnan for the generous gift of the GalT plasmid, S. Whiteheart for the OGA antibody, T.C. Neo for assistance with synthesis of ketogalactose probe 1, and A. Su for technical discussions. This work was supported by the US National Institutes of Health (RO1 NS045061), the National Science Foundation CAREER Award (CHE-0239861) and the Parson's Foundation (N.K.).

Author information



Corresponding author

Correspondence to Linda C Hsieh-Wilson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression levels of EGR-1, GRASP55 and eIF4G following kainic acid treatment of rats. (PDF 22 kb)

Supplementary Fig. 2

Annotated CAD MS4 and ETD MS/MS mass spectra for all sequenced peptides. (PDF 1955 kb)

Supplementary Table 1

Mean ratios of individual peptides from α-crystallin and OGT, and mean ratios of all peptides. (PDF 85 kb)

Supplementary Table 2

Identification and quantification of changes in O-GlcNAc glycosylation induced by kainic acid. (PDF 9 kb)

Supplementary Table 3

O-GlcNAc glycosylated proteins identified from the cerebral cortex of kainic acid-stimulated rats. (PDF 10 kb)

Supplementary Methods (PDF 151 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Khidekel, N., Ficarro, S., Clark, P. et al. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol 3, 339–348 (2007).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing