METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们正在开发分析结合蛋白质伙伴的硫酸乙酰肝素(HS)序列的方法。在本工作中，抗凝血酶III被用作HS结合蛋白的模型。方法是制备大小分级的HS寡糖，在生理条件下将它们与蛋白质结合(等渗盐)。将非结合低聚糖透析掉，并分析结合的低聚糖。变性步骤对这些过程至关重要。传统的方法使用高浓度的氯化钠来扰乱碳水化合物-蛋白质的相互作用。我们正在测试完成这一任务的质谱学友好方法，包括酸性pH、碱性pH和有机溶剂。HS在细胞表面、基底膜和细胞外基质中表达，由GlcNAc？1-4HexA？/？1-4的线性重复单元组成，硫化残基和糖醛酸异构体的排列不同。虽然已知HS的蛋白质结合作用是由特定的寡糖序列介导的，但结构鉴定方法远远落后于蛋白质组学方法。研究HS相互作用的经典技术包括使用具有离散特异性的肝素裂解酶结合层析或电泳分离来分析酶产物。本文研究了利用尺寸排阻色谱(SEC)LC-MS分析不同HS裂解酶样品和拆分酶抗性区域的方法。分离和纯化的HS链用肝素裂解酶I、II和III的组合进行解聚。利用紫外分光光度法监测每个反应的程度，以选择合适的时间点进行MS分析。在这些时间点用LC/MS分析低聚糖产物，使用Superdex多肽3.2/30秒色谱柱，用30%甲醇、0.1%氢氧化铵和Bruker Esquire 3000 ITMS(由Bruker Daltonics，Inc.捐赠)平衡。在负极模式下运行。采用L 30~100m/min的流速，以1650m/z/s的速度扫描离子，获得了最佳的分辨率。

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.