METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 硫酸乙酰肝素 (HS) 是一种线性多硫酸化多糖，定位于细胞表面和细胞外基质。这种多糖通过调节多种蛋白质的活性（包括凝血酶、生长因子和形态发生素）发挥重要的生物学作用。 HS 上的几种蛋白质结合表位已通过酶消化和二糖分析等传统方法进行了表征。在这里，我们描述了一种基于质谱的新型蛋白质结合表位表征方法（HS）。我们已将其应用于能够结合抗凝血酶 III (ATIII) 的八糖。 这些代表了迄今为止通过在线 LC/MS2 研究的最大的硫酸乙酰肝素寡糖。 通过用肝素裂解酶 III 彻底消化，然后在制备型尺寸排阻色谱 (SEC) 柱上对消化材料进行分级分离，生成猪肠粘膜 HS 八糖文库。为了纯化结合 ATIII 的八糖，将八糖 (dp8) SEC 级分与 ATIII 混合，并使用 SEC 将未结合的糖与糖-蛋白质复合物分离。随后，将复合物捕获在 C-18 柱上，并用 2M 乙酸铵洗脱结合的寡糖。 HS 八糖的表征是通过 ThermoFisher Scientific LTQ-Orbitrap 上的 LC/MS2 进行的，在使用内部填充的毛细管 amide-80 柱（TOSOH bioscience）（250um X 15 cm）和 Advion Triversa Nanomate 接口进行质谱检测之前进行在线色谱分离。

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 linear polysulfated polysaccharide that is localized to the cell surface and extracellular matrix. This polysaccharide plays a significant biological role by modulating the activities of a wide array of proteins including coagulation enzymes, growth factors and morphogens. Several protein binding epitopes on HS have been characterized by traditional methods such as enzymatic digestion and disaccharide analysis. Here we describe a novel mass spectrometric based approach for characterization approach (HS) of protein binding epitopes. We have applied it to octasaccharides capable of binding Antithrombin III (ATIII). These represent the largest heparan sulfate oligosaccharides studied by on-line LC/MS2 to date. Porcine intestinal mucosa HS octasaccharide libraries were generated by exhaustive digestion with heparin lyase III followed by fractionation of the digestion material on preparative size exclusion chromatography (SEC) column. In order to purify the octasaccharides that bind ATIII, the octasaccharide (dp8) SEC fraction was mixed with ATIII and the unbound sugars were separated from the sugar-protein complex using SEC. Subsequently, the complex was trapped on a C-18 cartridge and the bound oligosaccharides eluted with 2M ammonium acetate. The characterization of the HS octasaccharide was done by LC/MS2 on a ThermoFisher Scientific LTQ-Orbitrap with on-line chromatographic separation prior to mass spectrometric detection using a capillary amide-80 column (TOSOH bioscience) packed in-house (250um X 15 cm) and an Advion Triversa Nanomate interface.