Proteoglycans (PGs) and their glycosaminoglycan (GAG) chains play a prominent role in a biological system. PGs are among the most structurally complex of the glycoconjugates and are involved in signaling and cell-cell interaction. PGs contain a core protein to which one or more glycosaminoglycan chain is attached through an O-glycosidic linkage to specific serine residues in the core protein. Due to their complex and diverse structures, a major limitation has been the lack of the advanced analytical platforms for complex glycans. Decades of analytical and biochemical research have led to the routine sequencing and analysis of nucleic acids and proteins in both academic and clinical laboratories using highly sensitive analytical platforms. However, the development of similar platform technologies in glycoscience is at least 20 years behind nucleic acid and protein science. Dr. Linhardt’s laboratory was the first to apply a number of sophisticated analytical methods, including high-resolution electrophoresis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry, developed for nucleic acid and protein analysis, to the structure analysis of GAGs.
For structural analysis, recombinant heparin lyases are used to break the glycosaminoglycan chains into disaccharide/oligosaccharides. Different disaccharides/oligosaccharides can be separated by LC/CE and detected MS/LIF. Their structures can be solved using sophisticated NMR and MS/MS analysis. New MS methods include FT-ICR-MS, with CID, ECD and EDD based fragmentation methods.
Using a combination of separation and high-resolution mass spectrometric methods, for the first time, we can sequence the simplest glycosaminoglycan chains from bikunin (results were published in Nature Chemical Biology, Oct, 2011).
Glycosaminoglycans (GAGs) are functionally important in the developmental biology of multicellular organisms and their local concentrations in a healthy organism are well regulated. While the determination of GAGs, present at low concentrations, in biological fluids poses a difficult challenge. Dr. Linhardt’s laboratory developed a rapid and sensitive method by liquid chromatography−tandem mass spectrometry to efficiently determine low concentrations of glycosaminoglycans in human urine. This method allows the analysis of glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10- to 100- fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current study offers a method that is sufficiently sensitive to study changes in urinary GAG levels and compositions resulting from diseases outside of MPS such as inflammatory reactions and cancers.
Heparin, a widely used clinical anticoagulant, its analysis has become increasingly important since the contamination of the heparin supply chain in 2007-8. In recent years, Heparin-based drug products are becoming more dependent on mass spectrometry (MS) for high sensitivity, high-throughput analyses. Electrophoresis, offering extremely high-resolution separations, is particularly ideal for online coupling to MS. However, the negative-mode CE–MS interface, which is suitable and applicable to online analysis of negatively charged heparins, remains a major technical challenge. Dr. Linhardt’s group established a novel hyphenated CE-MS method relying on a reverse polarity separation with the analyte injected at the cathode and a coated sheath capillary interface to promote the electrokinetic pumping of negatively charged heparin oligosaccharides for MS analysis. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins.