Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O-GlcNAc Subproteome Upon Oxidative Stress

O-GlcNAc is thought to regulate proteins in a mannaer analogous to other PTMs, modulating cellular functions including the cellular stress response. The aim of this study was to identify specific cellular networks and protein complexes that are differentially O-GlcNAcylated and/or expressed upon acute oxidative stress (H2O2 for 1 and 2 h). We achieved this by employing SILAC and a novel anti-O-GlcNAc G5 lectibody IP strategy that combind O-GlcNAc specific antibodies and the lectin WGA. We identified 990 proteins among all three treatments including 202 non-redundant O-GlcNAc modified peptides. Differentially expressed proteins clustered into canonical 14-3-3/PI3K signaling pathways including the 14-3-3 complex, chaperonins, RNA Pol II Mediator and nuclear pore complexes.