New Chromatographic Technologies for Resolving Carbohydrate Isomers

 描述（由申请人提供）：从生物来源中分离复杂混合物高度依赖于微柱（毛细管）液相色谱（LC）的进展。当与生物分子质谱（MS）结合使用时，毛细管LC柱已经成为关键的“组学”技术方法的核心，包括在寻找疾病生物标志物中的生物液体和组织的糖蛋白质组学和糖组学分析。虽然分析实践中使用的典型色谱柱使用毛细管内填充的5 µ m大小的颗粒，但更多动力学渐进色谱柱类型现在可以紧密填充1.0- 1.5 µm范围内的颗粒。以较高的入口压力和优化柱填充技术所需的时间为代价，填充有这种小颗粒的毛细管可以明显优于当今蛋白质组学和代谢组学实践中使用的更传统的柱（如已经用反相分离所示）。然而，糖科学的分离问题需要不同的柱材料有效地保留相对亲水的碳水化合物。印第安纳州大学最近开发了非常有前途的新柱材料，并进行了初步测试，以满足这些需求：（1）大孔二氧化硅微球，这是通过超声喷雾热解（USP）制备，使用无机盐作为可移动的孔模板;和（2）大孔碳微球，使用有机羧酸盐，其USP产生形态上明确的实体。该提案涉及颗粒表面的适当改性，以利于吸附色谱和亲水相互作用色谱（HILIC）的开发，并提供迄今为止尚未实现的多种聚糖异构体的总分辨率。这是通过柱选择性和动力学性能的最佳组合来实现的。这项提案涉及到私家侦探的专业知识，一位生物分析化学家和分离科学专家，以及首席I。专攻材料化学这项研究还将得到北卡罗来纳州大学一位杰出合作者的大力协助， 另外两名合作者在碳水化合物合成方面具有重要的专业知识。目的1涉及大孔二氧化硅微粒的改性，用于在HILIC分离模式中保留极性糖缀合物溶质。将USP衍生的大孔二氧化硅颗粒填充到不同长度和直径的毛细管中，并在不同的压力和不同的移动的相下进行评价。将特别考虑生物学上重要的岩藻糖基化和唾液酸化异构体结构。在填充二氧化硅材料之后，柱将被原位处理以产生极性表面结构。目标2将处理填充不同类型的颗粒，特别是含碳材料。它们将接受与目标1类似的评价。这项研究将解决当代糖生物学和生物医学领域的迫切需求之一。

 DESCRIPTION (provided by applicant): Separation of complex mixtures from biological sources has been highly dependent on advances in microcolumn (capillary) liquid chromatography (LC). When used in combination with biomolecular mass spectrometry (MS), capillary LC columns have been at the heart of key "omics" technological approaches including glycoproteomic and glycomic profiling of biological fluids and tissues in search for disease biomarkers. While typical columns used in the analytical practice utilize 5 µm-sized particles packed inside capillary tubes, more kinetically progressive column types can now be tightly packed with particles within 1.0- 1.5 µm range. At the expense of higher inlet pressures and the needed time for optimizing column packing technologies, the capillaries packed with such small particles can clearly outperform the more conventional columns used in today's practice of proteomics and metabolomics (as it has been already shown with reversed-phase separations). However, the separation problems of the glycoscience need different column materials effectively retaining relatively hydrophilic carbohydrates. Highly promising new column materials have been recently developed at Indiana University and preliminarily tested to meet such needs: (1) macroporous silica microspheres, which are prepared by ultrasonic spray pyrolysis (USP) using inorganic salts as a removable pore template; and (2) macroporous carbon microspheres, employing organic carboxylate salts, whose USP yields morphologically well-defined entities. This proposal deals with appropriate modifications of the particle surfaces for the benefits of adsorption chromatography and hydrophilic interaction chromatography (HILIC) developments and toward providing thus far unachieved total resolution of numerous glycan isomers. This is to be achieved through the optimum combination of column selectivity and kinetic performance. The proposal involves a combination of expertise by the P.I., a bioanalytical chemist and a separation science specialist, and the co-I. specializing in materials chemistry. The study will also be significantly aided by a distinguished collaborator at the University of North Carolina and two other collaborators with significant expertise in carbohydrate synthesis. Aim 1 concerns modification of macroporous silica microparticles for retention of polar glycoconjugate solutes in the HILIC separation mode. The USP-derived macroporous silica particles will be packed into capillaries of different lengths and diameters and evaluated under different regimes of ultrahigh pressure and different mobile phases. Special consideration will be given to biologically important fucosylated and sialylated isomeric structures. After packing silica materials, the column will be treated in situ to generate polar surface structures. Aim 2 will deal with packing different types of particles, specifically carbonaceous materials. They will be subjected to simila evaluations as in Aim 1. The proposed research will address one of the urgent needs of contemporary glycobiology and biomedical field.