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。 糖胺聚糖(GAG)、肝素、硫酸肝素、皮肤素和硫酸软骨素参与重要的生理和病理过程，包括通过与细胞内和细胞间蛋白的直接相互作用来调节酶和信号分子，以响应细胞损伤(损伤、感染、肿瘤发生)、细菌感染等。在过去的十年里，越来越多的蛋白质被发现与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，我们建立了一种改进的基于LC-MS的结合分析方法。在这项工作中，使用基于酰胺的正相色谱柱的LC-MS方法被用来鉴定介导抗凝血酶III结合的肝素结构。 用肝素裂解酶-I在优化条件下对肝素进行消化，以实现部分消化。然后使用制备性尺寸排除色层(SEC)对低聚糖进行分级。聚合度(DP)分别为6、8、10的糖分别与ATIII混合。用高效SEC将游离糖从络合物中分离出来。将该络合物应用于反相柱，结合的寡糖用2M醋酸铵洗脱。然后用LC-MS对洗脱部分进行表征，色谱柱为250m×15 cm的TSK-Gel酰胺-80色谱柱。流动相：流动相：95%A：5%B(A=95%ACN，B=10%ACN，50 mM甲酸铵pH 4.4)，流速1？L/min。低聚糖以5%~75%B的梯度在60min内直接洗脱到工作在负离子模式下的QSTAR Pulsar-I质谱仪中。 毛细管正相LC/MS提供了灵敏和可重复性的数据，使结合或未结合的肝素低聚糖的表征变得容易。在目前的LC/MS系统中，肝素dp6的拆分可以达到30pmoL。检测到如下结构：[6][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]。DP6组分化合物的洗脱时间取决于硫酸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的完全拆分明确地表明，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通过硫酸盐损失而产生的。此外，还检测到3种糖型[1，2，3，6，1]。对于dp8和dp10组分也观察到了相同类型的结果。 同样的肝素DP6混合物受到先前描述的ATIII结合方案的影响。在预结合dp6混合物中检测到的所有结构中，从蛋白质洗脱后只检测到两个结合体，即[1，2，3，8，0]和[1，2，3，9，0]。除了这两个物种克服SEC柱和三轮洗涤的非平衡条件的能力外，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，ATIII通过加热灭活使其生物失活，然后与肝素dp6混合。在第二个实验中，将活性蛋白与非特异性低聚糖硫酸软骨素B混合。在两个实验中，都没有检测到低聚糖，证实了优化后的方法能够消除非特异性结合。这项工作将扩大到dp8和dp10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.