METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III

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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. We are developing methods for the analysis of heparan sulfate (HS) sequences that bind protein partners. In the present work, the antithrombin III is used as a model HS binding protein. The approach is to prepare size fractionated HS oligosaccharides, to bind them to a protein under physiological conditions (isotonic salt). Non-binding oligosaccharides are dialyzed away, and the bound oligosaccharides analyzed. The denaturation step is critical to these procedures. Traditional approaches use high sodium chloride concentrations to disrupt the carbohydrate-protein interactions. We are testing mass spectrometry-friendly approaches for accomplishing this task, including acidic pH, basic pH, and organic solvents. HS is expressed on cell surfaces, in basement membranes and extracellular matrices and is comprised of linear repeating units of GlcNAc?1-4HexA?/?1-4 with variations in the arrangement of sulfated residues and uronic acid epimers. Although it is known that the protein binding interactions of HS are mediated by specific oligosaccharide sequences, methods for structural elucidation lag far behind those used for proteomics. Classical techniques for studying HS interactions involve using heparin lyases with discrete specificities combined with chromatographic or electrophoretic separations for analyzing the enzyme products. The use of size exclusion chromatography (SEC) LC-MS for analysis of different HS lyase digestion samples and resolution of enzyme resistant regions is investigated in this work. Isolated and purified HS chains were subjected to depolymerization using combinations of heparin lyases I, II, and III. The extent of each reaction was monitored using UV spectrophotometry to select appropriate time points for MS analysis. Oligosaccharide products at these time points were analyzed by LC/MS using a Superdex peptide 3.2/30 SEC column, equilibrated with 30% methanol, 0.1% ammonium hydroxide and a Bruker Esquire 3000 ITMS (donated by Bruker Daltonics, Inc.) operating in the negative mode. Optimal resolution was achieved by utilizing flow rates of 30-100 ?l/min and ions were scanned at a rate of 1650 m/z per sec.