title: 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.
authors.affiliation: Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Nelly
authors.initials: N
authors.lastname: Khidekel
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.forename: Peter M
authors.initials: PM
authors.lastname: Clark
authors.forename: Marian C
authors.initials: MC
authors.lastname: Bryan
authors.forename: Danielle L
authors.initials: DL
authors.lastname: Swaney
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Rexach
authors.forename: Yi E
authors.initials: YE
authors.lastname: Sun
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: Eric C
authors.initials: EC
authors.lastname: Peters
authors.forename: Linda C
authors.initials: LC
authors.lastname: Hsieh-Wilson
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002236:Carbohydrate Conformation; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006031:Glycosylation; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020543:Proteome
chemical_list: D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D020543:Proteome; D000117:Acetylglucosamine