Article abstract
Nature Chemical Biology 3, 339 - 348 (2007)
Published online: 13 May 2007 | doi:10.1038/nchembio881
Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics
Nelly Khidekel1, Scott B Ficarro2, Peter M Clark1, Marian C Bryan1, Danielle L Swaney3, Jessica E Rexach4, Yi E Sun4, Joshua J Coon3, Eric C Peters2 & Linda C Hsieh-Wilson1
Abstract
The addition of the monosaccharide 
-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.
- Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
- Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA.
- Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
- Mental Retardation Research Center, Department of Psychiatry and Biobehavioral Sciences and Department of Molecular and Medical Pharmacology, and Neuropsychiatric Institute, The David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
Correspondence to: Linda C Hsieh-Wilson1 e-mail: lhw@caltech.edu
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