A PLATFORM FOR GAG DISACCHARIDE ANALYSIS USING SEC LC/MS

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structurally-polydisperse heparan sulfate (HS) chains are responsible for the interactions of HS proteoglycans with a wide variety of proteins, playing critical roles in biology. One HS biosynthesis step, the coordinated N-deacetylation/ N-sulfation on the GlcNAc residues, can be interrupted by the limited supply of 3'-phosphoadenosine 5'-phosphosulfate, and result in the formation of unsubstituted glucosamine residues within HS chains. Although endogenous N-unsubstituted saccharides have only been identified in a handful of studies, they have been implicated to be important in cellular and pathophysiological phenomena. Therefore, there is a clear need to systematically study the occurrence, and determine the structure and distribution patterns of these free-amino-containing HS disaccharides from mammals in a tissue-specific manner. HS from rat tissues was isolated by pronase and benzonase digest of lypholized tissue samples. HS was recovered by DEAE weak anion exchange procedure as part of the GAG pool. Both rat GAGs and bovine HS were exhaustively digested by heparin lyases and subject to SEC (Superdex Peptide column) LC/MS analysis. SEC LC/MS/MS was employed to illustrate the detailed structure of disaccharide isomers. Intact HS was N-acetylated by acetic anhydride-d6 and subjected to SEC LC/MS analysis. HS was also cleaved by nitrous acid at pH 4, with and without derivatizaion of the anhydromannose, before lyase digest and SEC LC/MS. An Applied Biosystem QSTAR Pulsar mass spectrometer equipped with TurboIonSpray interface was employed in this study.