MONOSACCHARIDE COMPOSITION ANALYSIS OF RELEASED N-LINKED GLYCANS 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。 在100 ℃下，用400 µ L 2.0 N三氟乙酸（TFA）水解释放的N-连接聚糖等分试样4 h。 水解后，将消化物在氮气流下干燥，重悬于H2O中，在冰中超声处理5 min，并转移至注射小瓶中。 以与样品相同的方式和同时水解具有已知摩尔数的中性糖和氨基糖的标准品的混合物。 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分析单糖。 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。 梯度程序使用以下移动的相洗脱液：脱气的纳米纯水和200 mM NaOH。 每45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. An aliquot of released N-linked glycans was hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 h. After hydrolysis, the digest was dried under a stream of nitrogen gas, resuspended in H2O, sonicated for 5 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars 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 monosaccharides 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 were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient program used the following mobile phase eluents: degassed nanopure water and 200 mM NaOH. Injection was made every 45 minutes. 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).