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： 取自2个埃彭多夫试管的样品合并到一个螺帽玻璃管中，并在氮气中干燥。然后用300000毫微米的L水重溶样品；150 L被输送到另一个螺帽玻璃管中进行唾液酸分析，其余的150 L被分配用于中性糖和氨基糖的分析。两份等分在氮气气流下干燥。中性糖和氨基糖分析用2N三氟乙酸在100oC下水解4h，唾液酸用2M冰醋酸在80oC下水解3h。所有的水解物都被冷冻干燥，再悬浮在水中，在冰中超声5min，转移到注射瓶中。 中性糖和氨基糖以及N-乙酰神经氨酸和N-羟基神经氨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以建立校正方程。样品中每个分析物的摩尔数通过校正方程的线性插值法进行量化。 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。单糖残留量用Dionex CarboPac PA20(3×150 mm)分析柱和氨基捕捉器分离。 在2对不同的流动相淋洗液和梯度程序中分析中性糖、氨基糖和唾液酸。流动相为纳米纯水和200 mM氢氧化钠(中性糖和氨基糖)和100 mM氢氧化钠(100 mM氢氧化钠和1M醋酸钠)和100 mM氢氧化钠(含100 mM氢氧化钠)。中性糖和氨基糖每45min注射一次，唾液酸分析每40min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: The samples from 2 eppendorf tubes were combined into a screw-cap glass tube and dried under N2. The sample was redissolved thereafter with 300 ¿L of nanopure water; 150 ¿L was pipetted into another screw-cap glass tube for sialic acids analysis and the remainder (150 ¿L) was allocated for neutral and amino sugars analysis. Both aliquots were dried under a stream of nitrogen gas. The 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 lyophilized thereafter, resuspended in H2O, sonicated for 5 min in ice and transferred to injection vials. A 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 sample. Four concentrations of the standard mixtures were prepared to establish calibration equations. The number of moles of each analyte 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 monosaccharide residues were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The neutral and amino sugars, and sialic acids were analyzed in 2 different pairs of mobile phase eluents and gradient programs. The mobile phase eluents were degassed nanopure water and 200 mM NaOH for neutral and amino sugars, and 100 mM NaOH and 1M sodium acetate in 100 mM NaOH for sialic acids. 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).