Chelsea Desbiens Graduate Student, Department of Chemistry University of Georgia Learn more about the speaker Wednesday, April 14, 2021 - 11:30am 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: Orlando, R. et al. IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J. Proteome Res. 2009, 8, 3816–3823. 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. 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. 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. Ma, J.; Hart, G. W., O-GlcNAc profiling: from proteins to proteomes. Clin. Proteomics 2014, 11 (1), 1–16.