MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

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： 将样品在其原始容器中冻干，用纳米纯H2O溶解，并将每个样品等分为2等份。 一份等分试样指定用于中性和氨基糖分析，另一份等分试样分配用于唾液酸分析。 将所有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用400 <$L 2.0 N三氟乙酸（TFA）在100 <$C下水解4 h，而用于唾液酸分析的等分试样用400 <$L 2.0 M乙酸在80 <$C下水解3 h。 将所有水解产物冻干，重悬于H2O中，在冰中超声处理7分钟，并转移至注射小瓶中。 以与样品相同的方式和同时水解中性糖和氨基糖以及具有已知摩尔数的唾液酸的标准品的混合物。 制备四种浓度的标准混合物以建立校准方程。 样品中每种残留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）分析柱分离单个中性糖和氨基糖以及唾液酸。 梯度程序使用以下移动的相洗脱液：A -脱气的纳米纯水和B -中性和氨基糖的200 mM NaOH，和C - 100 mM NaOH和D -唾液酸的100 mM NaOH中的1 M乙酸钠。 每45分钟注射中性糖和氨基糖，每40分钟注射唾液酸。 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）。

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 were lyophilized in their original containers, dissolved with nanopure H2O and each sample was divided equally into 2 aliquots. One aliquot was designated for neutral and amino sugars analysis with the other aliquot allocated for sialic acids analysis. All aliquots were dried under a stream of nitrogen gas. The aliquots intended for neutral and amino sugars analysis were hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 h, whereas the aliquots for sialic acids analysis were hydrolyzed with 400 ¿L of 2.0 M acetic acid at 80¿C for 3 h. All 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 samples. Four concentrations of standard mixture 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 and sialic acids were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used the following mobile phase 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 45 minutes for neutral and amino sugars and every 40 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).