MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

通过 HPAEC 进行单糖成分分析

• 批准号: 8170785
• 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/8170785
• 关键词: Aliquot; Amino Sugars; Anions; 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; Protocols documentation; Pump; Research; Research Personnel; Resources; Sampling; Sodium Acetate; Source; System; Time; Trifluoroacetic Acid; United States National Institutes of Health; Vial device; Water; base; data acquisition; detector; instrument; mannose 6 phosphate; 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. The sample was divided into two aliquots: one for neutral and amino sugars analysis and the other aliquot for mannose-6-phosphate analysis. The aliquot intended for neutral and amino sugars analysis was hydrolyzed with 2 N trifluoroacetic acid (TFA) at 100oC for 4 hours and the aliquot for mannose-6-phosphate analysis was hydrolyzed with 6.75 N TFA at 100oC for 1.5 hours. The hydrolysates were then dried under N2, redissolved 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 mannose-6-phosphate with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four concentration of the neutral and amino sugar standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) and mannose-6-phosphate (640, 1280, 2560, 5120 picomoles 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 mannose-6-phosphate 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 mannose-6-phosphate 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, 1 M sodium acetate in 100 mM NaOH for mannose-6-phosphate. Injections were made every 40 minutes for neutral and amino sugar determination and every 35 minutes for mannose-6-phosphate determination. 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 chromeleon software.

该副本是利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，这不一定是调查员的机构。 样品分为两个等分试样：一个用于中性和氨基糖分析，另一个用于甘露糖-6-磷酸分析的等分试样。 用于中性和氨基糖分析的等分试样在100OC下用2 N三氟乙酸（TFA）水解4小时，而将甘露糖-6-磷酸分析的等分试样用6.75 N TFA在100oC中水解1.5小时。 然后将水解液在N2下干燥，在H2O中重新溶解，在冰中进行超声检查，并转移到注射小瓶中。 与中性糖和氨基糖的标准组合，以及具有已知数量痣的甘露糖 - 6-磷酸盐的混合物的混合物与样品的相同和同时水解了。 准备四个浓度的中性和氨基糖标准混合物（每次注射0.5、1.0、2.0和4.0 nmoles）和6-磷酸甘露糖（640、1280、2560、5120 picomoles每注射）以建立校准方程。 样品中每糖的摩尔数量通过校准方程的线性插值来量化。 使用配备有梯度泵，电化学检测器和自动进样器的Dionex ICS3000系统，通过HPAEC分析了中性和氨基糖和6-磷酸甘露糖。 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甘露糖-6-磷酸盐分离。 梯度程序使用脱位A，脱气的纳米水和B，200 mM NaOH用于中性和氨基糖，以及C，100 mM NaOH和D，100 mM NaOH中的1 M乙酸钠，用于100 mm NaOH，用于甘露糖-6-磷酸盐。 每40分钟进行一次注射以进行中性和氨基糖的测定，每35分钟进行甘露糖 - 6-磷酸盐的测定。 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 Chromeleon软件完成了仪器控制和数据采集。