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 (

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…This chromatography technique employs low flow rates and an isocratic elution buffer containing low concentrations of GlcNAc (Vosseller et al 2006). LWAC has been adapted successfully for shotgun characterization of O-GlcNAcmodified proteomes from postsynaptic density preparations (Vosseller et al 2006), Arabidposis thaliana (Xu et al 2012), and mouse embryonic stem cells (Myers et al 2011). …”

“…Wheat germ agglutinin (WGA) is a commonly used plant lectin for enriching and detecting O-GlcNAc-modified proteins (Zachara et al 2004a(Zachara et al , 2011bVosseller et al 2006). WGA has weak affinity for single GlcNAc residues, but its affinity is dramatically increased for GlcNAcs that are clustered (Finlay et al 1987;Lundquist and Toone 2002;Lee and Lee 2000).…”

“…Lectin weak affinity chromatography (LWAC) utilizes the affinity of WGA in an extended column format (>3 m in length) with a small diameter (∼1 cm) to enrich O-GlcNAc-modified peptides (Vosseller et al 2006). This chromatography technique employs low flow rates and an isocratic elution buffer containing low concentrations of GlcNAc (Vosseller et al 2006).…”

“…Examples of O-GlcNAcylated proteins include chromatinassociated proteins and histones, transcription factors, ribosomal proteins, proteasomal proteins, cytoskeletal proteins, and many different types of signaling proteins such as kinases and metabolic enzymes (Carapito et al 2009;Gurcel et al 2008;Rexach et al 2008;Teo et al 2010;Vosseller et al 2006;Wang et al 2010a, b;Wells et al 2002b). O-GlcNAc is thought to regulate these proteins in a manner analogous to protein phosphorylation.…”

“…Introduction O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) is a dynamic post-translational modification (PTM) of more than 3,000 nuclear, cytoplasmic, and mitochondrial proteins (Carapito et al 2009;Gurcel et al 2008;Rexach et al 2008;Teo et al 2010;Vosseller et al 2006;Wang et al 2010a, b;Wells et al 2002b;Torres and Hart 1984). While O-GlcNAc appears more common in metazoans, there is evidence for this PTM in simple eukaryotes such as Aspergillus niger (Machida and Jigami 1994) and in some prokaryotes (Shen et al 2006).…”