O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.

TitleO-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.
Publication TypeJournal Article
Year of Publication2006
AuthorsVosseller K, Trinidad JC, Chalkley RJ, Specht CG, Thalhammer A, Lynn AJ, Snedecor JO, Guan S, Medzihradszky KF, Maltby DA, Schoepfer R, Burlingame AL
JournalMol Cell Proteomics
Volume5
Issue5
Pagination923-34
Date Published2006 May
ISSN1535-9476
KeywordsAcetylglucosamine, Amino Acid Sequence, Animals, Cattle, Chromatography, Affinity, Lectins, Mass Spectrometry, Mice, Molecular Sequence Data, Peptides, Proteomics, Synapses
Abstract

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. Beta-elimination/Michael addition, MS(3) 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.

DOI10.1074/mcp.T500040-MCP200
PubMed URLhttp://www.ncbi.nlm.nih.gov/pubmed/16452088?dopt=Abstract
Alternate TitleMol. Cell Proteomics
PubMed ID16452088