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

Abstract

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.

Disaccharide Analysis of Glycosaminoglycans Using Hydrophilic Interaction Chromatography and Mass Spectrometry

“…In a previous study, we had ascertained the identities of the HONO-derived disaccharides using PGC-MS. 23 Since no HexA-aMan standards are available commercially, this PGC-MS method was used to purify each deaminative cleavage-derived disaccharide. These purified disaccharides were analyzed by HILIC to establish their retention times (Supporting Figure S-7).…”

“…In recent years, liquid chromatography-mass spectrometry (LC-MS) has emerged as the tool of choice for GAG disaccharide analysis. 23–28 The unique elution times of the disaccharides together with the m/z values provide sufficient information to assign each disaccharide. In addition, the use of tandem MS provides structural information on peak identities.…”

Pharmacology of Heparin and Related Drugs

“…This method of depolymerization has the great disadvantage that both iduronate and glucuronate yield the same unsaturated residue at the nonreducing end; information on the nonreducing terminal uronic acid is therefore lost. Techniques for separation and mass spectrometric characterization of nitrous acid depolymerized oligosaccharides, with intact uronic acids at the nonreducing ends, have been devised (Gill et al, 2012). Combined techniques, including a separation step and a mass spectrometry (MS) step, are powerful (Zaia, 2009).…”

Glycosaminoglycan Glycomics Using Mass Spectrometry

“…Such methods range from the direct infusion of lyase disaccharides (26 -28) to a variety of LC/MS systems using chromatography methods including reversed phase (29 -31), reversed-phase ion pairing (32)(33)(34), size exclusion (18), porous graphitized carbon (51, 52) and hydrophilic interaction. Recently, an LC/MS method for the analysis of deaminative cleavage disaccharides was published (35). MS has the advantage of being able to detect low-abundance products in the digestion mixtures, including saturated disaccharides from the non-reducing polysaccharide termini and rare 3-O-sulfated disaccharides.…”