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Molecular & Cellular Proteomics 5:923-934, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

O-Linked N-Acetylglucosamine Proteomics of Postsynaptic Density Preparations Using Lectin Weak Affinity Chromatography and Mass Spectrometry^*,S

Keith Vosseller^,, Jonathan C. Trinidad, Robert J. Chalkley, Christian G. Specht^¶, Agnes Thalhammer^¶, Aenoch J. Lynn, June O. Snedecor, Shenheng Guan, Katalin F. Medzihradszky, David A. Maltby, Ralf Schoepfer^¶ and Alma L. Burlingame^,||

From the Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 and ^¶ Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. ß-Elimination/Michael addition, MS³ on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.

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