Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics
Abstract
The addition of the monosaccharide beta-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.
Additional Information
© 2007 Nature Publishing Group. Received 21 December 2006; accepted 13 April 2007; published online 13 May 2007. 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 Compound 1Compound 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.).Attached Files
Supplemental Material - nchembio881-S1.pdf
Supplemental Material - nchembio881-S2.pdf
Supplemental Material - nchembio881-S3.pdf
Supplemental Material - nchembio881-S4.pdf
Supplemental Material - nchembio881-S5.pdf
Supplemental Material - nchembio881-S6.pdf
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Additional details
- Eprint ID
- 56470
- Resolver ID
- CaltechAUTHORS:20150408-095425960
- RO1 NS045061
- NIH
- CHE-0239861
- NSF
- Ralph M. Parsons Foundation
- Created
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2015-04-08Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field