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. Heparan sulfate (HS) is a polyanionic, linear polysaccharide that plays a role in many biological functions by modulating the binding activity of extracellular ligands. It is comprised of alternating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) units and has the general formula [GlcA?(1,4)GlcNAc?(1,4)]n. This structure may be modified by epimerization of GlcA to iduronic acid and by sulfation of this residue at the 2O- position. The structure can then be 3O-, 6O-, or N-sulfated on GlcN. With a total of 48 possible disaccharide structures, HS represents the most highly variable biological macromolecule. Structural analysis of HS chains is complicated due to the inherent heterogeneity of this compound class. The work presented herein extends the knowledge of HS domain structure through the analysis of tissue-derived HS oligosaccharides by LC/MS. Commercially available bovine kidney HS (Sigma) as well as purified bovine HS from aorta, lung, intestine, and kidney was completely depolymerized with heparin lyases. The resultant disaccharides were derivatized with 2-aminoacridone and analyzed by capillary electrophoresis (CE). For oligosaccharide analysis, HS samples were digested to partial completion. The products of each digestion were reductively aminated with custom tetraplex stable isotope tags to facilitate analysis of three sample replicates in one LC/MS run. Samples were injected onto a 250 ?m x 15 cm capillary column packed in-house with Amide-80 resin (Tosoh) connected online to an Applied Biosystems/MDS Sciex QSTAR mass spectrometer or a Thermo Electron Corporation LTQ Orbitrap mass spectrometer. Glycan compositions were assigned using a Microsoft Excel based database and abundances calculated based on integrated peak area in the MS mode. The goal of this work was to determine how HS structures vary as a function of organ derivation. From CE analysis of exhaustive lyase digests, it was found that HS from different bovine organs shows distinct differences in disaccharide composition. Specifically, the degree to which the HS chains are sulfated or acetylated differs between organ types. While important, disaccharide analysis only begins to provide a structural understanding of the role of HS in protein-carbohydrate interactions. Larger oligosaccharides must be generated in order to investigate intact HS domain structures, which consist of N-sulfated and N-acetylated regions. In order to better understand this domain organization and to optimize conditions for analysis, multiple lyase digestions of bovine kidney HS were produced (including 20, 40, 60, 80, and 100% digestion completion). Oligosaccharides ranging from dimers to octamers could be simultaneously analyzed by the amide LC/MS system. Based on this analysis, an appropriate partial digest will be selected in order to analyze oligosaccharides derived from the various available bovine organ HS samples. The analysis will provide an HS oligosaccharide profile that is a function of organ type.
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