MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 批准号: 8363058
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363058
• 关键词: Acetic Acids; Aliquot; Amino Sugars; Anions; Calibration; Carbohydrates; Chromatography; Equation; Funding; Gases; Glycoproteins; Grant; Ice; Individual; Injection of therapeutic agent; Methanol; Methods; Mole the mammal; Monosaccharides; National Center for Research Resources; Nitrogen; Phase; Principal Investigator; Protocols documentation; Pump; Research; Research Infrastructure; Resources; Sampling; Sialic Acids; Sodium Acetate; Source; Stream; System; Technology; Time; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Vial device; Water; base; cost; detector; programs

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Each sample was dissolved with 400 ¿L of nanopure H2O , and divided equally into two aliquots and transferred into pre-rinsed (with methanol) screw-cap tubes. One aliquot was designated for neutral and amino sugars analysis with the other aliquot allocated for sialic acids analysis. All aliquots were dried under a stream of nitrogen gas. The aliquots intended for neutral and amino sugars analysis were hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 h, whereas the aliquots for sialic acids analysis were hydrolyzed with 400 ¿L of 2.0 M acetic acid at 80¿C for 3 h. The hydrolysates were lyophilized, resuspended in H2O, sonicated for 7 min in ice and transferred to injection vials. A mix of standards for neutral and amino sugars, and for sialic acids with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. Four concentrations of standard mixture were prepared to establish a calibration equation. The number of moles of each residue in the sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars, and sialic acids were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The individual neutral and amino sugars and sialic acids were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used the following mobile phase eluents: A - degassed nanopure water and B - 200 mM NaOH for neutral and amino sugars, and C - 100 mM NaOH and D - 1M sodium acetate in 100 mM NaOH for sialic acids. Injections were made every 45 minutes for neutral and amino sugars and every 40 min for sialic acids. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates", 1994, Methods Enzymol. 230: 208-225).

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 方法： 每个样品用 400 µL 纳米纯 H2O 溶解，并等分为两等份，并转移到预冲洗（用甲醇）的旋盖管中。 一份指定用于中性糖和氨基糖分析，另一份指定用于唾液酸分析。 所有等分试样在氮气流下干燥。 用于中性和氨基糖分析的等分试样用 400 L 2.0 N 三氟乙酸 (TFA) 在 100°C 下水解 4 小时，而用于唾液酸分析的等分试样则用 400 L 2.0 M 乙酸在 80°C 下水解 3 小时。 将水解产物冻干，重悬于水中，在冰中超声处理 7 分钟，然后转移至注射瓶中。 以与样品相同的方式和同时水解中性糖和氨基糖以及已知摩尔数唾液酸的标准混合物。 制备四种浓度的标准混合物以建立校准方程。 通过校准方程的线性插值来量化样品中每种残留物的摩尔数。 使用配备梯度泵、电化学检测器和自动进样器的 Dionex ICS3000 系统，通过 HPAEC 分析中性糖、氨基糖和唾液酸。 通过带有氨基捕集器的 Dionex CarboPac PA20 (3 x 150 mm) 分析柱分离各个中性糖、氨基糖和唾液酸。 梯度程序使用以下流动相洗脱液：A - 脱气纳米纯水和 B - 200 mM NaOH（用于中性糖和氨基糖），C - 100 mM NaOH 和 D - 1M 乙酸钠（溶于 100 mM NaOH）用于唾液酸。 中性糖和氨基糖每 45 分钟注射一次，唾液酸每 40 分钟注射一次。 所有方法均基于Hardy和Townsend描述的方案（Hardy，M.R.和Townsend，R.R.，“糖蛋白衍生碳水化合物的高pH阴离子交换层析”，1994，Methods Enzymol.230：208-225）。