MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 批准号: 8363073
• 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/8363073
• 关键词: Acetic Acids; Aliquot; Amino Sugars; Anions; Calibration; Carbohydrates; Chromatography; Equation; Funding; Gases; Glycoproteins; Grant; Ice; Individual; Injection of therapeutic agent; 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; 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: The samples were lyophilized in their original containers, dissolved with nanopure H2O and each sample was divided equally into 2 aliquots. 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. All 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).

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 方法： 样品在其原始容器中冻干，用纳米H2O溶解，并将每个样品平均分为2个等分试样。一个等分试样用于中性和氨基糖分析，另一种分配的等分试样用于唾液酸分析。所有等分试样均在氮气流下干燥。将用于中性和氨基糖分析的等分试样用400°L的2.0 N三氟乙酸（TFA）在100°C时持续4小时，而唾液酸分析的等分试样用400°c乙酸分析的等分试样在80°C下的400°l含2.0 m乙酸。将所有水解物均冻干，在H2O中恢复，在冰中超声超声，并转移到注射小瓶中。 中性和氨基糖的标准含量的混合物，以及具有已知数量分子的唾液酸的混合物，以与样品相同的方式和同时水解。准备四个浓度的标准混合物来建立校准方程。通过校准方程中的线性插值来量化样品中每个住所的分子数量。 使用配备有梯度泵，电化学检测器和自动采样器的Dionex ICS3000系统，通过HPAEC分析了中性和氨基糖以及唾液酸。用氨基陷阱的Dionex Carbopac PA20（3 x 150 mm）分析柱将单个中性和氨基糖和唾液酸分离。梯度程序使用以下流动相位洗脱液：A-脱水的纳米水和B -200 mM NaOH用于中性和氨基糖，以及100 mm NaOH中的100 mm NaOH和D -1M乙酸钠，用于唾液酸。每45分钟进行每45分钟的注射剂，以进行中性和氨基糖，每40分钟唾液酸。所有方法均基于Hardy和Townsend所描述的方案（Hardy，M。R.和Townsend，R。R.，“糖蛋白衍生的碳水化合物的高ph阴离子 - 交换色谱”，1994年，方法酶。230：208-225）。