Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics.
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.
Khidekel
Nelly
N
Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Ficarro
Scott B
SB
Clark
Peter M
PM
Bryan
Marian C
MC
Swaney
Danielle L
DL
Rexach
Jessica E
JE
Sun
Yi E
YE
Coon
Joshua J
JJ
Peters
Eric C
EC
Hsieh-Wilson
Linda C
LC
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
D006023:Glycoproteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D020543:Proteome; D000117:Acetylglucosamine