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