MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 批准号: 8363083
• 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/8363083
• 关键词: Acetic Acids; Aliquot; Amino Sugars; Anions; Calibration; Carbohydrates; Chromatography; Equation; Funding; Gases; Glycoproteins; Grant; Hydrolysis; Ice; Injection of therapeutic agent; Methods; Mole the mammal; Monosaccharides; N-Acetylneuraminic Acid; N-glycolylneuraminic acid; National Center for Research Resources; Nitrogen; Phase; Principal Investigator; Protocols documentation; Pump; Research; Research Infrastructure; Resources; Running; Saliva; Sampling; Sialic Acids; Sodium Acetate; Solutions; Source; System; Technology; Time; Trifluoroacetic Acid; 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: A 1-mL solution of the NANA sample with a concentration of 0.03 ¿g/¿L was prepared and was used for analysis of N-acetylneuraminic acid content. The NANA sample was not subjected to any hydrolysis step. For the SALIVA sample, two 200-¿g aliquots were prepared for neutral and amino sugars, and sialic acids analyses. The saliva aliquot for neutral and amino sugars analysis was hydrolyzed with 2N trifluoroacetic acid at 100oC for 4 hr, whereas the aliquot for sialic acids analysis was hydrolyzed with 2M acetic acid at 80oC for 3 hr. All hydrolysates were dried under nitrogen gas thereafter, resuspended in H2O, sonicated for 5 min in ice and transferred to injection vials. A mix of unhydrolyzed standards for N-acetylneuraminic acid and N-glycolylneuraminic acid was run at the same time as the NANA sample. Another mix of standards for neutral and amino sugars, and N-acetylneuraminic acid and N-glycolylneuraminic acid were hydrolyzed in the same manner and at the same time as the SALIVA sample. Four concentrations of the standard mixtures were prepared to establish calibration equations. The number of moles of each analyte in the NANA or SALIVA 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 monosaccharide residues 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 analysis. Injections were made every 45 min for neutral and amino sugars and every 40 min for sialic acids analysis. 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 该子项目的主要支持。 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 来源的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： 浓度为 0.03 ¿ 的 1 mL NANA 样品溶液克/克L 被制备并用于分析 NANA 样品，对于 SALIVA 样品，两个 200-¿为中性糖和氨基糖制备g等份，用于中性糖和氨基糖分析的唾液酸等份用2N三氟乙酸在100℃下水解4小时，而用于唾液酸分析的等份用2M乙酸在20℃下水解。 80℃ 3 小时，然后将所有水解产物在氮气下干燥，重悬于水中，在冰中超声处理 5 分钟并转移至注射瓶中。 N-乙酰神经氨酸和N-乙醇酰神经氨酸的未水解标准品混合物与NANA样品同时运行。中性糖和氨基糖以及N-乙酰神经氨酸和N-乙醇酰神经氨酸的另一种标准品混合物在样品中被水解。以与唾液样品相同的方式同时制备四种浓度的标准混合物以建立校准方程。 NANA 或 SALIVA 样品中的分析物通过校准方程的线性插值进行定量。 使用配有梯度泵、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。单糖残留物通过 Dionex CarboPac PA20 (3 x 150 mm) 分析柱进行分离。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 乙酸钠用于唾液酸分析，每 45 分钟注射一次用于中性糖和氨基糖，每 40 分钟注射一次用于唾液酸分析。方法基于 Hardy 和 Townsend（Hardy, M. R. 和 Townsend, R. R.，“高 pH 阴离子交换糖蛋白衍生碳水化合物的色谱法”，1994，Methods Enzymol.230：208-225）。