MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 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
• 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/8363094
• 关键词: Acetic Acids; Aliquot; Amino Sugars; Anions; Calibration; Carbohydrates; Chromatography; Equation; Funding; Gases; Glass; Glycoproteins; Grant; 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; 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: 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).

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 将2个eppendorf管中的样品合并到螺旋盖玻璃管中，并在N2下干燥。 此后，用300 μ L纳米纯水重新溶解样品;将150 μ L移液到另一个螺旋盖玻璃管中用于唾液酸分析，剩余部分（150 μ L）用于中性糖和氨基糖分析。 将两份等分试样在氮气流下干燥。 用于中性糖和氨基糖分析的等分试样在100 ℃下用2N三氟乙酸水解4小时，而用于唾液酸分析的等分试样在80 ℃下用2 M乙酸水解3小时。 以与样品相同的方式和同时水解中性糖和氨基糖以及N-乙酰神经氨酸和N-羟乙酰神经氨酸的标准品混合物。 制备四种浓度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进行定量。 使用配备有梯度泵、电化学检测器和自动进样器的Dionex ICS 3000系统，通过HPAEC分析中性和氨基糖以及唾液酸。 通过具有氨基捕集器的Dionex CarboPac PA 20（3 X 150 mm）分析柱分离单糖残基。 在2对不同的移动的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，移动的相洗脱液为脱气的纳米纯水和200 mM NaOH，对于唾液酸，为100 mM NaOH和1 M乙酸钠的100 mM NaOH溶液。 每45分钟注射中性糖和氨基糖，每40分钟注射唾液酸分析。 所有方法均基于哈代和汤森（哈代，M. R.，和汤森，R. R.，“糖蛋白衍生碳水化合物的高pH阴离子交换色谱法”，1994，酶学方法。230：208-225）。