Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress

O-Linked N-acetyl-β-d-glucos­amine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcN­Acylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcN­Acylated upon acute oxidative stress (H2O2) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel “G5-lectibody” immuno­precipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic reversed phase chromatography and C18 RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins that were identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcN­Acylation status of 24 proteins. Analysis of proteins with altered glycosylation suggests that stress-induced changes in O-GlcN­Acylation cluster into pathways known to regulate the cell’s response to injury and include protein folding, transcriptional regulation, epi­genetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcN­Acylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.