PROJECT SUMMARY/ABSTRACT Sulfated glycosaminoglycan (GAG) carbohydrates represent one of the more structurally diverse groups of biomolecules, and a comprehensive understanding of their biological structure-function relationships has yet to be achieved. Unlike other biomolecules such as DNA/RNA and proteins that are synthesized based upon a template, GAG biosynthesis is the result of the cumulative actions of a series of enzymes to produce a dynamic, polydisperse mixture. The composition of this mixture is dependent on factors such as organism age, developmental or disease state and tissue of origin. Although this diversity presents a daunting analytical challenge, significant progress has been made in the field through attempts to isolate and characterize GAGs ranging from intact polysaccharides to enzymatically prepared oligosaccharides and disaccharides. The most widespread approach is to profile GAG disaccharides via separation (HPLC, UHPLC, HILIC, CE) and detection (UV, fluorescence, mass spectrometry (MS)). Domains can be characterized for structure- function studies by combining these techniques into hyphenated methods (e.g., LC-MS). Even though disaccharide analysis is the most widely utilized method for GAG analysis, there are no readily available sources of internal standards for MS-based quantitation. Recently, multiple reaction monitoring (MRM) has been increasingly applied to GAG analysis and internal standards would significantly enhance the quantitative nature of such approaches. We propose to leverage the biosynthetic machinery of CHO-S cells, which are widely employed in the production of protein pharmaceuticals and are known to produce GAGs, as a means to generate GAGs containing stable isotopes, currently named isoGAGs. Our approach will utilize 13C6 D-glucose and an in vivo method to introduce 15N into the UDP-sugar intermediates that form the backbone of the GAG chain. As these are stable isotopes, they do not add any safety concerns. Initial efforts will be focused on the creation of a series of heparan sulfate (HS) disaccharides that we envision as a commercially available library for quantitation during disaccharide profiling experiments.

PROJECT NARRATIVE The accurate quantitation of carbohydrates (sp. glycosaminoglycans, GAGs) is a standing analytical need in the characterization of structure-function relationships as well as biomedical applications. Currently, there is no commercial source of reference standards for the GAG community to serve as internal standards in quantitation efforts that cover all representative disaccharide units. This proposal will develop a robust method to biosynthesize isotopically labeled heparan sulfate (HS) GAG disaccharides via CHO-S cells which are widespread in use for the production of protein pharmaceuticals and are known to produce GAGs in measurable quantity.