MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 每个样品用400°L纳米水溶解，等分成两份，转移到预冲洗(含甲醇)的螺纹帽试管中。一个等分用于中性糖和氨基糖的分析，另一个等分用于唾液酸的分析。所有等分在氮气气流下干燥。用于中性糖和氨基糖分析的等价物在100℃下用2.0N三氟乙酸(TFA)400°L水解4h，用于唾液酸分析的等价物在80℃用2.0M冰醋酸400°L水解液3h，水解物冷冻干燥，在水中悬浮，在冰中超声7min，转移到注射瓶中。 中性糖和氨基糖以及已知摩尔数的唾液酸的标准混合物以与样品相同的方式和同时进行水解。制备了四种浓度的标准混合物，建立了校正方程。样品中每个残留物的摩尔数由校正方程中的线性插值法定量。 中性糖、氨基糖和唾液酸的分析采用Dionex ICS3000系统，该系统配备了梯度泵、电化学检测器和自动进样器。中性糖、氨基糖和唾液酸用Dionex CarboPac PA20(3×150 mm)色谱柱和氨基捕捉器分离。梯度程序使用以下流动相洗脱剂：A-脱气纳米纯水和B-200 mM氢氧化钠用于中性和氨基糖，C-100 mM氢氧化钠和D-1M醋酸钠在100 mM氢氧化钠中用于唾液酸。中性糖和氨基糖每45分钟注射一次，唾液酸每40分钟注射一次。所有方法都基于Hardy和Townsen(Hardy，M.R.和Townend，R.，“High-pH阴离子交换色层析糖蛋白衍生碳水化合物”，1994，Methods Enzymol)所描述的方案。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: Each sample was dissolved with 400 ¿L of nanopure H2O , and divided equally into two aliquots and transferred into pre-rinsed (with methanol) screw-cap tubes. 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. 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 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).