Hydrogen/deuterium exchange-LC-MS approach to characterize the action of heparan sulfate C5-epimerase

• Published in: Analytical and bioanalytical chemistry 2011-07, Vol.401 (1), p.237-244
• Main Authors: Babu, Ponnusamy, Victor, Xylophone V., Nelsen, Emily, Nguyen, Thao Kim Nu, Raman, Karthik, Kuberan, Balagurunathan
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Animals; Anticoagulants (Medicine); Assaying; Biochemistry; Carbohydrate Epimerases - metabolism; Cell Line; Chains; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatographic methods and physical methods associated with chromatography; Chromatography, Liquid - methods; Deuterium; Deuterium Exchange Measurement - methods; Disaccharides - isolation & purification; Enzymes; Exact sciences and technology; Food Science; Glycosaminoglycans; Heparin - metabolism; Heparitin Sulfate - metabolism; High performance liquid chromatography; Hydrogen; Insecta - enzymology; Laboratory Medicine; Mass Spectrometry - methods; Mathematical analysis; Monitoring/Environmental Analysis; Original Paper; Other chromatographic methods; Polysaccharides; Precursors; Proteoglycans - metabolism; Residues; Spectrometric and optical methods; Sugars; Sulfates
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-011-5087-z

Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on HS precursors, converts glucuronyl residues into iduronyl residues, and modulates subsequent biosynthetic steps in vivo. Previously, the ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA- A Man R and IdoA- A Man R disaccharides using a tedious paper chromatography technique. This radioactive assay, based on measuring either the release or incorporation of 3 H at C5 carbon of uronyl residues of 3 H-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA- A Man R and IdoA- A Man R disaccharides on high-performance liquid chromatography in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive-based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reactions on the same substrate catalyzed by C5-epimerase. The results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi. Figure Using hydrogen/deuterium exchange, Heparan Sulfate C5-Epimerase activity is characterized using a novel DEEP-LC-MS method