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Abstract
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-Gln. Here, we present an adaptation of this method, Dynamic IDAWG, that allows for the calculation of half-lives of released glycans in a given sample following analyses by mass spectrometry. Simultaneously, the amount of sialic acid recycling for an identified glycan can also be calculated. An additional benefit to using this method is that the 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 processes. O-GlcNAc is thought to be a dynamic modification; that is, the modification exhibits a shorter turnover rate than the modified protein. 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 turnover rate of O-GlcNAc on nuclear and cytosolic proteins.