Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications

Abstract

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked ␤-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild ␤-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using ␤-elimination/Michael addition methods. The rapid identification of proteins by mass spectrometry has become commonplace in the postgenomic era (1). However, one major challenge that remains is the identification of post-translational modifications on these proteins. More than 25 years ago, Finn Wold and colleagues (2) recognized the abundance of naturally occurring modified forms of the genetically encoded 21 amino acids. In addition to phosphorylation, a variety of post-translational modifications, including acetylation (3), methylation (4), and O-linked ␤-N-acetylglucosamine (OGlcNAc) 1 (5-7), are now recognized to regulate protein functions in cellular processes. Therefore, identification of proteins along with their post-translational modifications, which has been referred to as "functional proteomics," is an important step in the characterization of proteomes. O-GlcNAc is a dynamic post-translational modification occurring on a variety of nucleocytoplasmic proteins and, in several instances, O-GlcNAc maps to the same or adjacent sites as phosphorylation (8, 9). Diverse classes of proteins are modified including cytoskeletal proteins, transcription factors, signaling adapter molecules, hormone receptors, nuclear pore complex (NPC) proteins, and kinases (10). The nucleocytoplasmic enzymes for the addition (O-GlcNAc transferase) and removal (neutral ␤-N-acetylglucosaminidase, O-GlcNAcase) of this modification have been cloned and characterized (11-16) and may ac...

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

“…To prevent labeling of phosphorylation sites, peptides were treated with CIP (0.4 U/ml in 100 mM NaCl, 10 mM MgCl 2 , 50 mM NH 4 HCO 3 , pH 8) at 37˚C for 4 h followed by desalting. To label O-GlcNAc sites, peptides were incubated in BEMAD buffer (1% triethylamine, 0.1% NaOH) for 2 h at 56˚C in the presence of 20 mM light (d0) DTT or heavy (d6) DTT (1,4-DTT-1,1,2,3,4,4-d6, CDN Isotopes) as described previously (41,42). The labeling reaction was quenched with 1% trifluoroacetic acid and the heavy and light labeling reactions were combined into a single aliquot for desalting.…”

“…Similar results were obtained with two additional donors across two independent experiments using anti-O-GlcNAc Ab CTD110. 6. involves alkaline cleavage of the sugar from peptides with subsequent addition of a DTT adduct (41,42). Unlike the native O-GlcNAc linkage, which is labile and tends to undergo neutral loss from the peptide backbone, the DTT adduct remains stable during conventional collision-induced dissociation mass spectrometry, allowing detection of the modification site.…”

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

“…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).…”

Mass spectrometry in aging research

“…However, it should be noted that b-elimination is not restricted to phosphoamino acids. For example, O-linked b-N-acetylglucosamine (O-GlcNAQc)-modified serine or threonine suffer b-elimination, on which basis Wells et al developed a procedure termed mild b-elimination followed by Michael addition with dithiothreitol (BEMAD) for the selective mapping of O-GlcNAc sites (Wells et al, 2002). In an alternative approach, phosphopeptides can be selectively enriched using immobilized metal affinity chromatography (IMAC) on supports containing trivalent metal ions such as Fe 3þ or Ga 3þ (Zarling et al, 2000;Riggs, Sioma, & Regnier, 2001;Chen et al, 2002;Ficarro et al, 2002).…”