Open AccessCombined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress
Author(s) -
Albert Lee,
Devin Miller,
Roger Henry,
Venkata D P Paruchuri,
Robert N. O’Meally,
Tatiana Boronina,
Robert N. Cole,
Natasha E. Zachara
Publication year - 2016
Publication title -
journal of proteome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.644
H-Index - 161
eISSN - 1535-3907
pISSN - 1535-3893
DOI - 10.1021/acs.jproteome.6b00369
Subject(s) - stable isotope labeling by amino acids in cell culture , oxidative stress , stress granule , biochemistry , immunoprecipitation , proteome , proteomics , microbiology and biotechnology , biogenesis , biology , glycosylation , lectin , cell , chemistry , translation (biology) , gene , messenger rna
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 (H 2 O 2 ) 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 C 18 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.