# Isotopically Labeled Heparan Sulfate Glycosaminoglycan Disaccharides for use as Internal Standards

> **NIH NIH R41** · GLYCOSCIENTIFIC, LLC · 2020 · $252,114

## Abstract

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.

## Key facts

- **NIH application ID:** 10080563
- **Project number:** 1R41GM139440-01
- **Recipient organization:** GLYCOSCIENTIFIC, LLC
- **Principal Investigator:** RON ORLANDO
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $252,114
- **Award type:** 1
- **Project period:** 2020-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10080563

## Citation

> US National Institutes of Health, RePORTER application 10080563, Isotopically Labeled Heparan Sulfate Glycosaminoglycan Disaccharides for use as Internal Standards (1R41GM139440-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10080563. Licensed CC0.

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