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： 制备1 mL浓度为0.03 μ g/L的NANA样品溶液，用于分析N-乙酰神经氨酸含量。 NANA样品未进行任何水解步骤。 对于SALIVA样品，制备两份200 μ g等分试样，用于中性糖和氨基糖以及唾液酸分析。 用于中性糖和氨基糖分析的唾液等分试样在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。 N-乙酰神经氨酸和N-羟乙酰神经氨酸的未水解标准品混合物与NANA样品同时运行。 以与SALIVA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-羟乙酰神经氨酸的另一种标准品混合物。 制备四种浓度的标准混合物以建立校准方程。 NANA或SALIVA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。 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，200 mM NaOH用于中性和氨基糖;和C，100 mM NaOH，和D，100 mM NaOH中的1 M乙酸钠用于唾液酸分析。 中性糖和氨基糖每45分钟注射一次，唾液酸分析每40分钟注射一次。 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: A 1-mL solution of the NANA sample with a concentration of 0.03 ¿g/¿L was prepared and was used for analysis of N-acetylneuraminic acid content. The NANA sample was not subjected to any hydrolysis step. For the SALIVA sample, two 200-¿g aliquots were prepared for neutral and amino sugars, and sialic acids analyses. The saliva 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 dried under nitrogen gas thereafter, resuspended in H2O, sonicated for 5 min in ice and transferred to injection vials. A mix of unhydrolyzed standards for N-acetylneuraminic acid and N-glycolylneuraminic acid was run at the same time as the NANA sample. Another 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 SALIVA sample. Four concentrations of the standard mixtures were prepared to establish calibration equations. The number of moles of each analyte in the NANA or SALIVA 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 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 analysis. 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).