The post-translational modification, O-linked β-N-acetylglucosamine (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. A single gene encodes the enzyme O-GlcNAc-transferase (OGT) allowing for the addition of O-GlcNAc onto Serine and Threonine residues, while another gene encodes for O-GlcNAc-hydrolase (OGA), which facilitates the removal of the aforementioned modification. It is hypothesized that O-GlcNAc is a dynamic modification; that is, the modification exhibits a shorter half-life than the modified protein.  Furthermore, it is believed that dynamics vary among different modification sites as well as the proteins that are modified and that levels, localization, and kinetics of the cycling influence this property. Unfortunately, dynamics has only been evaluated on a small number of O-GlcNAc modified proteins due to the laborious and insensitive methods utilized. Therefore, there is an urgent need in the field to develop high-throughput, sensitive methods to evaluate the dynamics of O-GlcNAc and O-GlcNAc modified proteins.<br/>To address this gap in knowledge, a novel methodology is under development in our group. The method known as Dynamic Isotopic Detection of Aminosugars With Glutamine (IDAWG) was originally designed as a quantification tool for glycomics to look at released glycans labeled with amide-<sup>15</sup>N-Gln taking advantage of the hexosamine biosynthetic pathway. Here, we present preliminary data using the dynamic IDAWG approach coupled with innovative enrichment and mass spectrometry techniques to look at turnover of O-GlcNAc modified glycopeptides. The culmination of these techniques should allow for examination of the dynamic nature of the O-GlcNAc modification and the modified proteins to be observed specifically in any cell line.

Defining O-GlcNAc cycling rates utilizing dynamic isotopic detection of amino sugars with glutamine (IDAWG)

2019 Fall National Meeting

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