METHODS FOR DETERMINATION OF HS EPITOPES THAT BIND ANTITHROMBIN III

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 糖胺聚糖（GAG）;肝素、硫酸乙酰肝素、皮肤素和硫酸软骨素参与重要的生理学和病理学过程，包括通过与细胞内和细胞间蛋白质的直接相互作用，对细胞损伤（创伤、感染、肿瘤发生）、细菌感染等作出反应的酶和信号分子的调节。在过去的十年中，已经发现越来越多的蛋白质与GAG相互作用。 为了鉴定这些蛋白质以及了解这些相互作用和与GAG-蛋白质相互作用相关的结构基序，我们开发了一种改进的基于LC-MS的结合测定方法。在这项工作中，使用基于酰胺的正相色谱柱的LC-MS方法用于鉴定介导抗凝血酶III结合的肝素结构。 在优化条件下用肝素裂解酶-I消化肝素，以实现部分消化。 然后使用制备型尺寸排阻色谱法（SEC）分级分离寡聚核苷酸。将聚合度（dp）为6、8、10的糖分别与ATIII混合。 使用高效SEC从复合物中分离未结合的糖。 将复合物施加到反相柱上，并用2 M乙酸铵洗脱结合的寡糖。 然后使用内部填充的250 μ m x 15 cm TSK-凝胶酰胺-80柱通过LC-MS表征洗脱的级分。在移动的相中以1 μ L/min用95%A：5%B（A=95%ACN，B=10%ACN，50 mM甲酸铵pH 4.4）平衡色谱柱。 用5%至75% B的梯度经60分钟将寡聚体直接洗脱到以负离子模式操作的QSTAR Pulsar-I质谱仪中。 毛细管正相LC/MS产生灵敏和可重复的数据，允许结合或未结合的肝素寡糖的简单表征。 使用当前的LC/MS系统，可以实现30 pmol肝素dp 6的非抗菌分离度。 检测到以下结构：[<$HexA，HexA，GlcN，SO 3，Ac] [1，2，3，6，1]，[1，2，3，7，1]，[1，2，3，7，0]，[1，2，3，8，0]，[1，2，3，9，0]。 dp 6馏分化合物的洗脱时间基于硫酸化基团与固定相的极性相互作用。 这些化合物在非重叠保留时间处的完全分离明确表明，每个结构[1，2，3，X，Y]不是通过[1，2，3，X+1，Y]结构的源内CID的硫酸盐损失产生的。 此外，还检测到3种糖型组成[1，2，3，6，1]。 对于dp 8和dp 10级分观察到相同类型的结果。 使肝素dp 6的相同混合物经受先前描述的ATIII结合方案。在预结合的dp 6混合物中检测到的所有结构中，在从蛋白质洗脱后仅检测到两种结合物，即[1，2，3，8，0]和[1，2，3，9，0]。除了这两种物质克服SEC柱的非平衡条件和三轮洗涤的能力之外，还用两个独立的实验进一步证实了它们的结合特异性。在第一个实验中，通过热灭活使ATIII失去生物活性，随后与肝素dp 6混合。在第二个实验中，将活性蛋白与非特异性寡糖硫酸软骨素B混合。在这两个实验中，处理后均未检测到寡糖，证实了优化方案消除非特异性结合的能力。 这项工作将扩展到dp 8和dp 10馏分。

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. Glycoaminoglycans (GAGs); heparin, heparan sulfate, dermatan and chondroitin sulfate are involved in important physiological and pathological processes including regulation of enzymes and signaling molecules in response to cellular damage (wounding, infection, tumorigenesis), bacterial infection, etc. via direct interaction with intra- and inter-cellular proteins. Over the past decade an increasing number of proteins have been discovered to interact with GAGs. In order to identify these proteins as well as understand these interactions and structural motifs related to GAG-protein interaction we developed an improved LC-MS based binding assay method. In this work, an LC-MS method using an amide-based normal phase chromatography column was used to identify heparin structures that mediate antithrombin III binding. Heparin was digested with heparin lyase-I under conditions optimized to achieve partial digestion. Oligosaccharides were then fractionated using preparative size exclusion chromatography (SEC). Sugars with degree of polymerization (dp) 6, 8, 10 were respectively mixed with ATIII. The unbound sugars were separated from the complex using high performance SEC. The complex was applied to a reverse phase cartridge and the bound oligosaccharides eluted with 2M ammonium acetate. The eluted fractions were then characterized by LC-MS using a 250 ¿m x 15 cm TSK-gel Amide-80 column packed in house. The column was equilibrated in mobile phase at 1 ¿L/min with 95%A:5%B (A=95%ACN, B=10%ACN, 50 mM ammonium formate pH 4.4. Oligosaccharides were eluted with a gradient of 5% to 75% B over 60 min directly into a QSTAR Pulsar-I mass spectrometer operating in the negative ion mode. Capillary normal phase LC/MS yielded sensitive and reproducible data allowing facile characterization of the bound or unbound heparin oligosaccharides. Unambiguous resolution of 30 pmol of heparin dp6 could be achieved using the current LC/MS system. The following structures were detected: [¿HexA, HexA, GlcN, SO3, Ac] [1,2,3,6,1], [1,2,3,7,1], [1,2,3,7,0], [1,2,3,8,0], [1,2,3,9,0]. The elution time of the dp6 fraction compounds is based on polar interactions of the sulfated group with the stationary phase. The complete resolution of those compounds at non-overlapping retention times demonstrate without any ambiguity that each structure [1,2,3,X,Y] is not generated by a sulfate loss through in-source CID of the [1,2,3,X+1,Y] structure. Furthermore, 3 glycoforms of composition [1,2,3,6,1] were detected. The same type of results was observed for the dp8 and dp10 fractions. The same mixture of heparin dp6 was subjected to the previously described ATIII binding protocol. Among all the structures detected in the pre-bound dp6 mixture, only two binders were detected after elution from the protein, namely [1,2,3,8,0] and [1,2,3,9,0]. In addition to the ability of these two species to overcome the non-equilibrium conditions of the SEC column and three rounds of washes, their binding specificity was further confirmed with two independent experiments. In the first experiment, the ATIII was rendered biologically inactive by heat inactivation and subsequently mixed with heparin dp6. In the second experiment, the active protein was mixed with the unspecific oligosaccharide Chondroitin sulfate B. In both experiments, no oligosaccharides were detected after the workup, confirming the ability of the optimized protocol to eliminate non-specific binding. The work will be expanded to the dp8 and dp10 fractions.