MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

基本信息

批准号：
8363094
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
项目类别：
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

项目摘要

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 from 2 eppendorf tubes were combined into a screw-cap glass tube and dried under N2. The sample was redissolved thereafter with 300 ¿L of nanopure water; 150 ¿L was pipetted into another screw-cap glass tube for sialic acids analysis and the remainder (150 ¿L) was allocated for neutral and amino sugars analysis. Both aliquots were dried under a stream of nitrogen gas. The 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 lyophilized thereafter, resuspended in H2O, sonicated for 5 min in ice and transferred to injection vials. A 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 sample. Four concentrations of the standard mixtures were prepared to establish calibration equations. The number of moles of each analyte 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 monosaccharide residues were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The neutral and amino sugars, and sialic acids were analyzed in 2 different pairs of mobile phase eluents and gradient programs. The mobile phase eluents were degassed nanopure water and 200 mM NaOH for neutral and amino sugars, and 100 mM NaOH and 1M sodium acetate in 100 mM NaOH for sialic acids. 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：将来自 2 个 eppendorf 管的样品合并到螺旋盖玻璃管中并在 N2 下干燥，然后用 300 ¿ 重新溶解样品。升纳米纯水；150 ¿将 L 移入另一个螺旋盖玻璃管中用于唾液酸分析，剩余的 (150 L) 用于中性糖和氨基糖分析。两份等分试样在氮气流下干燥。用 2N 三氟乙酸在 100℃ 水解 4 小时，而用于唾液酸分析的等分试样则用 2M 乙酸在 100℃ 水解。 80℃ 3 小时，然后将所有水解产物冻干，重悬于水中，在冰中超声处理 5 分钟，然后转移至注射瓶中。以与样品相同的方式同时水解中性糖和氨基糖以及N-乙酰神经氨酸和N-乙醇酰神经氨酸的标准混合物，以建立校准方程。样品中每种分析物的摩尔数通过校准方程的线性插值来量化。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。与氨基陷阱。在 2 对不同的流动相洗脱液和梯度程序中分析中性糖和氨基糖以及唾液酸，对于中性糖和氨基糖，流动相洗脱液为脱气纳米纯水和 200 mM NaOH，以及 100 mM NaOH 和 1M 乙酸钠。对于唾液酸，每 45 分钟注射一次 100 mM NaOH，对于唾液酸，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 阴离子交换色谱”，1994，Methods Enzymol. 230：208-225）。。