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 C18RPLC-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.
Department of Biological Chemistry, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
Albert
A
Lee
Department of Biological Chemistry, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
Devin
D
Miller
Department of Biological Chemistry, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
Roger
R
Henry
Department of Biological Chemistry, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
Venkata D P
VD
Paruchuri
Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205-2185, United States.
Robert N
RN
O'Meally
Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205-2185, United States.
Tatiana
T
Boronina
Department of Biological Chemistry, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.|Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine , 733 North Broadway Street, Baltimore, Maryland 21205-2185, United States.
Robert N
RN
Cole
Department of Biological Chemistry, The Johns Hopkins University School of Medicine , 725 North Wolfe Street, Baltimore, Maryland 21205-2185, United States.
Natasha E
NE
Zachara
D000117:Acetylglucosamine / Q000276:immunology / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D000906:Antibodies; D002470:Cell Survival; D056148:Chromatography, Reverse-Phase; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D007553:Isotope Labeling; D037102:Lectins / Q000276:immunology; D018384:Oxidative Stress / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis / Q000378:metabolism*
D000906:Antibodies; D037102:Lectins; D020543:Proteome; D006861:Hydrogen Peroxide; D000117:Acetylglucosamine
O-GlcNAc; glycoproteins; glycosylation; signal transduction
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