MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 批准号: 8170771
• 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/8170771
• 关键词: Acetic Acids; Aliquot; Amino Sugars; Anions; CD7 gene; Calibration; Carbohydrates; Chromatography; Computer Retrieval of Information on Scientific Projects Database; Computer software; Equation; Funding; Glycoproteins; Grant; Hour; Hydrolysis; Ice; Individual; Injection of therapeutic agent; Institution; Methods; Mole the mammal; Monosaccharides; Needles; Protocols documentation; Pump; Research; Research Personnel; Resources; Sampling; Sialic Acids; Sodium Acetate; Source; Stainless Steel; System; Time; Trifluoroacetic Acid; United States National Institutes of Health; Vial device; Water; base; data acquisition; detector; instrument; programs; sugar

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. Monosaccharide composition analysis by HPAEC The sample was divided equally into two aliquots (~375 ¿g each): one for neutral and amino sugars analysis and the other aliquot for sialic acid analysis. The aliquot intended for neutral and amino sugars analysis were hydrolyzed with 2 N trifluoroacetic acid at 100 oC for 4 hours and those for sialic acid analysis were hydrolyzed with 2 M acetic acid at 80oC for 3 hours. The hydrolysates were then lyophilized, redissoled in H2O, sonicated for 7 min in ice and transferred to an injection vial. 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 sugar 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 DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. 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 eluents A, degassed nanopure water; B, 200 mM NaOH; and C, 100 mM NaOH for the neutral and amino sugars; and D, 1 M sodium acetate in 100 mM NaOH for sialic acids. Injections were made every 40 minutes for neutral and amino sugar determinations and every 35 minutes for sialic acid determinations. 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). Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.

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。 HPAEC的单糖组成分析 将样品平均分为两个等分试样（〜375»g）：一个用于中性和氨基糖分析，另一个用于唾液酸分析。将用于中性和氨基糖分析的等分试样用2 N三氟乙酸以100 oC水解4小时，并在80oC下用2 M乙酸水解唾液酸分析3小时。然后将水解物冻干，在H2O中重新固定，在冰中超声超声处理，并转移到注射小瓶中。 与中性糖和氨基糖的标准混合在一起，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（每次注射0.5、1.0、2.0和4.0 Nmoles）以建立校准方程。样品中每种糖的分子数量通过校准方程的线性插值来量化。 使用配备有GP40梯度泵，ED40电化学探测器的DX500系统和氨基糖和唾液酸分析了HPAEC，以及含有不锈钢针的热散热AS3500 AutoSampler。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以及唾液酸分离。梯度程序使用了脱脂纳米水的洗脱液； B，200毫米NaOH；和C，中性和氨基糖的100毫米NaOH； d，100 mM NaOH中的1 m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的测定，每35分钟进行唾液酸的测定。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）。使用Dionex PeakNet软件（版本5.01）完成了仪器控制和数据采集。