Mass Spectrometric Method for Determining the Uronic Acid Epimerization in Heparan Sulfate Disaccharides Generated Using Nitrous Acid

Department of Biochemistry, Boston University School of Medicine , Boston, Massachusetts, United States.
Analytical Chemistry (Impact Factor: 5.64). 08/2012; 84(17):7539-46. DOI: 10.1021/ac3016054
Source: PubMed


Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography-mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin.

14 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the non-reducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography (HILIC)-MS for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC-MS platform.
    Analytical Chemistry 12/2012; 85(2). DOI:10.1021/ac3030448 · 5.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The fact that sulfated glycosaminoglycans (GAG) are necessary for functioning of all animal physiological systems drives the need to understand their biology. This understanding is limited, however, by the heterogeneous nature of GAG chains and their dynamic spatial and temporal expression patterns. GAGs have a regulated structure overlaid by heterogeneity but lack the details necessary to build structure/function relationships. In order to provide this information, we need glycomics platforms that are sensitive, robust, high throughput, and information-rich. This review summarizes progress on mass spectrometry-based GAG glycomics methods. The areas covered include disaccharide analysis, oligosaccharide profiling, and tandem mass spectrometric sequencing.
    Molecular &amp Cellular Proteomics 01/2013; 12(4). DOI:10.1074/mcp.R112.026294 · 6.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Biological functions of a variety of proteins are mediated via their interaction with glycosaminoglycans (GAGs). The structural diversity within the wide GAG landscape provides individual interaction sites for a multitude of proteins involved in several pathophysiological processes. This 'GAG angle' of such proteins as well as their specific GAG ligands give rise to novel therapeutic concepts for drug development. Current glycomic technologies to elucidate the glycan structure-function relationships, methods to investigate the selectivity and specificity of glycan-protein interactions and existing therapeutic approaches to interfere with GAG-protein interactions are discussed.
    Expert Review of Proteomics 02/2013; 10(1):77-95. DOI:10.1586/epr.12.75 · 2.90 Impact Factor
Show more