MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 批准号: 8170781
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170781
• 关键词: Acetic Acids; Aliquot; Amino Sugars; Anions; Calibration; Carbohydrates; Chromatography; Computer Retrieval of Information on Scientific Projects Database; Equation; Funding; Glycoproteins; Grant; Hydrolysis; Ice; Individual; Injection of therapeutic agent; Institution; Methods; Mole the mammal; Monosaccharides; Protocols documentation; Pump; Research; Research Personnel; Resources; Sampling; Sialic Acids; Sodium Acetate; Solutions; Source; System; Time; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Vial device; Water; base; detector; programs

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The sample was transferred from the original container to a screw cap tube, dried under N2,, redissolved with H2O and divided equally the solution into two aliquots, and lyophilized. One aliquot was designated for neutral and amino sugars analysis with the other aliquot for sialic acids analysis. The aliquot intended for neutral and amino sugars was hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 h, whereas the aliquot for sialic acids was hydrolyzed with400 ¿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 sample. Four concentration of standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) 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 were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used 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 40 minutes for neutral and amino sugars and every 35 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).

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 将样品从原始容器转移到螺帽管，在N2下干燥，用H2O重新溶解，并将溶液平均分为两个等分试样，并进行冻干。一个等分试样用于中性和氨基糖分析，另一种等分试样用于唾液酸分析。将用于中性和氨基糖的等分试样用400°L的2.0 n三氟乙酸（TFA）在100°C下于100 h持续4小时，而唾液酸的等分酸在80 h时将唾液酸的等分试样水解为400°c。将水解液冻干，在H2O中恢复，在冰中超声超声，并转移到注射小瓶中。 中性和氨基糖和具有已知数量分子数量的唾液酸的标准混合物以与样品相同的方式和同一时间水解。准备四个浓度的标准混合物（每次注射0.5、1.0、2.0和4.0 Nmoles）以建立校准方程。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分离。梯度程序使用脱胶纳米水和B -200 mM NaOH用于中性和氨基糖，以及100 mM NaOH和D -1M NaOH中的100 mM NaOH和D -1M乙酸钠用于唾液酸。每40分钟进行一次注射，以进行中性和氨基糖，每35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）。