New Chromatographic Technologies for Resolving Carbohydrate Isomers

解析碳水化合物异构体的新色谱技术

• 批准号: 8985005
• 负责人: Milos Novotny
• 金额: $ 31.6万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8985005
• 关键词: Address; Adsorption; Area; Biocompatible Materials; Biological; Biological Markers; Blood capillaries; Buffers; Caliber; Carbohydrates; Carbon; Chemicals; Chemistry; Chromatography; Collaborations; Complex; Complex Mixtures; Detection; Detergents; Development; Diagnostic Neoplasm Staging; Disease; Ensure; Equipment; Evaluation; Geometry; Glycobiology; Glycoconjugates; Glycopeptides; Glycoproteins; Heart; In Situ; Indiana; Isomerism; Kinetics; Laboratories; Lead; Lectin; Length; Liquid substance; Mass Spectrum Analysis; Materials Testing; Measurement; Medical; Methodology; Methods; Microspheres; Modeling; Modification; Monosaccharides; Morphology; North Carolina; Oligonucleotides; Oligosaccharides; Particle Size; Peptides; Performance; Phase; Physiological; Polymers; Polysaccharides; Procedures; Protein Glycosylation; Proteomics; Pump; Research; Research Personnel; Resolution; Salts; Science; Scientist; Silanes; Silicon Dioxide; Solvents; Source; Specialist; Structure; Surface; System; Techniques; Technology; Testing; Time; Tissues; Tube; Ultrasonics; Universities; Variant; Work; base; capillary; capillary liquid chromatography; carboxylate; human disease; instrument; interest; medical specialties; meetings; metabolomics; particle; physical property; pressure; professor; public health relevance; silane; solute; sugar; tandem mass spectrometry

 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.

 描述(申请人提供)：复杂混合物与生物来源的分离高度依赖于微柱(毛细管)液相色谱(LC)的进步。当与生物分子质谱仪(MS)结合使用时，毛细管液相色谱柱一直是关键的“组学”技术方法的核心，包括生物体液和组织的糖蛋白组学和糖谱分析，以寻找疾病生物标记物。虽然分析实践中使用的典型色谱柱使用填充在毛细管中的5微米大小的颗粒，但更动态的进样柱类型现在可以紧密填充1.0-1.5微米范围内的颗粒。以更高的进样压力和优化柱填充技术所需的时间为代价，填充了如此小颗粒的毛细管显然可以超越当今蛋白质组和代谢组学实践中使用的更传统的柱(正如反相分离已经表明的那样)。然而，糖学的分离问题需要不同的柱材料来有效地保留相对亲水性的碳水化合物。印第安纳大学最近开发了非常有前途的新型色谱柱材料，并对其进行了初步测试，以满足以下需求：(1)大孔二氧化硅微球，使用无机盐作为可移除的孔模板，通过超声喷雾热解(USP)制备；(2)大孔碳微球，使用有机羧酸盐，其USP产生形态明确的实体。这项建议涉及对颗粒表面进行适当的修饰，以利于吸附层析和亲水相互作用层析(HILIC)的发展，并朝着提供迄今为止尚未实现的大量糖链异构体的完全拆分的方向发展。这是通过柱选择性和动力学性能的最佳组合来实现的。该提案涉及私家侦探、一名生物分析化学家和一名分离科学专家，以及一名协理的专业知识。专攻材料化学。这项研究还将得到北卡罗来纳大学一位杰出的合作者的大力帮助， 另外两个在碳水化合物合成方面拥有丰富专业知识的合作者。目的1研究大孔二氧化硅微粒在HILIC分离模式下保留极性糖共轭溶质的改性。USP法制备的大孔二氧化硅颗粒将被填充到不同长度和直径的毛细管中，并在不同的超高压和不同的流动相下进行评估。将特别考虑具有生物重要性的岩藻糖化和唾液酸化异构体结构。填充硅胶材料后，柱将进行原位处理，以生成极性表面结构。目标2将处理不同类型的颗粒的包装，特别是碳质材料。他们将接受与目标1类似的评估。拟议的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。