METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 硫酸乙酰肝素(HS)是一种线性聚硫化多糖，定位于细胞表面和细胞外基质。这种多糖通过调节包括凝血酶、生长因子和形态因子在内的一系列蛋白质的活性，发挥着重要的生物学作用。用传统的酶消化和双糖分析等方法对HS上的几种蛋白质结合表位进行了表征。在这里，我们描述了一种新的基于质谱学的蛋白质结合表位表征方法。我们已经将其应用于能够结合抗凝血酶III(ATIII)的八糖。这是迄今为止通过在线LC/MS2研究的最大的硫酸乙酰肝素低聚糖。 用肝素裂解酶III对猪肠黏膜进行彻底消化，然后在制备性尺寸排除层析(SEC)柱上进行分级，得到猪肠黏膜HS八糖文库。为了纯化与ATIII结合的八糖，将八糖(Dp8)SEC组分与ATIII混合，用SEC从糖-蛋白质复合体中分离出未结合的糖。随后，络合物被捕获在C-18色谱柱上，结合的寡糖用2M醋酸铵洗脱。HS八糖的表征通过LC/MS2在ThermoFisher Science LTQ-Orbitrap上进行，在线色层分离，然后使用内部填充的毛细管酰胺-80柱(Tosoh Bioscience)(250微米×15厘米)和Advion Triversa Nanomate界面进行质谱分析。

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.