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Dynamic Isotopic Detection of Aminosugars With Glutamine (IDAWG) for Released Glycans and O-GlcNAc Modified Proteins

Chelsea Desbiens
Chelsea Desbiens
Graduate Student, Department of Chemistry
University of Georgia
ONLINE ONLY
Analytical Seminar

The Isotopic Detection of Aminosugars With Glutamine (IDAWG) method was originally developed for the glycomics field as a quantitative tool that takes advantage of the hexosamine biosynthetic pathway, isotopically labeling nitrogen-containing glycans in cell culture systems via the use of 15N-Gln1,2. Here, we present an adaptation of this method, Dynamic IDAWG, that allows for the calculation of half-lives and sialic acid remodeling for released glycans in a given sample following analyses by mass spectrometry. An additional benefit of this method is that cycling rates of the post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) can also be determined. O-GlcNAc is found on thousands of nuclear and cytosolic proteins in mammals and is thought to be a regulatory modification, playing a role in a variety of cellular processes3. The modification cycles on and off serine and threonine residues by means of O-GlcNAc Transferase (OGT) and O-GlcNAc Hydrolase (OGA)4. O-GlcNAc is thought to be a dynamic modification; that is, the modification exhibits a shorter half-life than the modified protein5.  However, dynamics have only been evaluated on a small number of O-GlcNAc modified proteins due to the laborious and insensitive methods that are available. Therefore, there is an urgent need in the field to develop high-throughput, sensitive methods to evaluate the dynamics of O-GlcNAc on a global scale. Here, we illustrate the utility of Dynamic IDAWG in cell culture systems to evaluate the turnover of complex N- and O-linked glycans as well as delineating the half-life of O-GlcNAc on nuclear and cytosolic proteins. 

References:

  1. Orlando, R. et al. IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J. Proteome Res. 2009, 8, 3816–3823.
  2. Fang, M.; Lim, J.-M.; Wells, L. Quantitative Glycomics of Cultured Cells Using Isotopic Detection of Aminosugars with Glutamine (IDAWG). Curr. Protoc. Chem. Biol. 2010, 2, 55–69.
  3. Torres, C.-R.; Hart, G. W., Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J. Biol. Chem. 1984, 259 (5), 3308–3317.
  4. Hart, G. W.; Slawson, C.; Ramirez-Correa, G.; Lagerlof, O., Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.  Annu. Rev. Biochem. 2011, 80 (1), 825–858.
  5. Ma, J.; Hart, G. W., O-GlcNAc profiling: from proteins to proteomes. Clin. Proteomics 2014, 11 (1), 1–16.

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