Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides

Abstract

Protein O-GlcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GlcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GlcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment-significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GlcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GlcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GlcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GlcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification. modification site assignment ͉ posttranslational modification ͉ proteomics ͉ Bassoon

Tandem mass spectrometry identifies many mouse brain O -GlcNAcylated proteins including EGF domain-specific O -GlcNAc transferase targets

“…Several methods to enrich O-GlcNAcylated proteins or peptides have led to the identification of a limited number of O-GlcNAcylation sites by MS. For example, lectin weak-affinity chromatography (4,10,14) enabled identification of up to 142 O-GlcNAcylation sites in 62 proteins from mouse embryonic stem cells (15). Immunoprecipitation, using O-GlcNAc-specific monoclonal antibodies (13,16), identified 83 O-GlcNAcylated sites from a HEK293T cell extract (13).…”

“…A recent metabolic labeling study that used alkyne-modified GlcNAc incorporated into OGT substrates and Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition (CuAAC) to a chemically cleavable biotin-azide probe, enabled identification of 374 putative O-GlcNAc modified proteins, but yielded no information on sites of O-GlcNAc modification (17). In addition to limited coverage of O-GlcNAcylation sites, a drawback to using these approaches is that they typically require milligram quantities of protein (4,10,13,14,16) or are limited to cultured cells (17), which makes them generally ill-suited for clinically derived tissue samples often available in small amounts.…”

“…Current understanding of the function of O-GlcNAcylation in neurodegeneration has been impeded by difficulties in identifying this modification, even using MS (10,11). First, the substoichiometric levels of O-GlcNAc modification at given sites on protein substrates necessitate enrichment of O-GlcNAcylated proteins or peptides before sequence analysis by MS (11).…”

Multi-Stage Mass Spectrometry Analysis of Sugar-Conjugated β-Turn Structures to be Used as Probes in Autoimmune Diseases

“…In fact, little evidence exists confirming the actual in vivo activity of these endoglycosydases. Chalkley et al [34] proposed that the identification of GlcNAc peptides in post-synaptic density preparations from murine brains might be due to an artifact during sample preparation.…”

The role of Ser-(Arg-Ser-Arg-Ser-GlucNAc)19-GlucNAc Fasciola gigantica glycoprotein in the diagnosis of prepatent fasciolosis in rabbits

“…High-sensitivity mass spectrometry (MS) provides means of directly addressing the both problems (Haslam et al 2001). Detailed structural analysis of carbohydrates is now possible through a combination of techniques including enzymatic or chemical degradation, separation by electrophoresis or chromatography and analysis using mass spectrometry (Dell 1987;Dell et al 1994;Haslam et al 2001;Hirabayashi and Kasai 2002;Chalkley et al, 2009). Moreover the potentials of glycoproteins (Ghosh et al 2005a, b;McAllister et al 2011;Georgieva et al 2012;Young et al 2012) or even glycolipids (Wuhrer et al 2003;Wuhrer et al 2004) in the diagnosis of fasciolosis are proved.…”