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