MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 将样品分成两等份：一等份用于中性和氨基糖分析，另一等份用于甘露糖-6-磷酸分析。 用于中性糖和氨基糖分析的等分试样用2 N三氟乙酸（TFA）在100 ℃下水解4小时，用于甘露糖-6-磷酸分析的等分试样用6.75 N TFA在100 ℃下水解1.5小时。 然后将水解产物在N2下干燥，再溶解于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甘露糖-6-磷酸的标准品的混合物。 制备四种浓度的中性和氨基糖标准混合物（每次注射0.5、1.0、2.0和4.0纳摩尔）和甘露糖-6-磷酸（每次注射640、1280、2560、5120皮摩尔），以建立校准方程。 样品中每种糖的摩尔数通过校准方程的线性插值来定量。 使用配有梯度泵、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甘露糖-6-磷酸。 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甘露糖-6-磷酸。 梯度程序使用洗脱液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乙酸钠用于甘露糖-6-磷酸。 每40分钟注射一次用于中性糖和氨基糖测定，每35分钟注射一次用于甘露糖-6-磷酸测定。 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）。仪器控制和数据采集使用Dionex chromatography on软件完成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The sample was divided into two aliquots: one for neutral and amino sugars analysis and the other aliquot for mannose-6-phosphate analysis. The aliquot intended for neutral and amino sugars analysis was hydrolyzed with 2 N trifluoroacetic acid (TFA) at 100oC for 4 hours and the aliquot for mannose-6-phosphate analysis was hydrolyzed with 6.75 N TFA at 100oC for 1.5 hours. The hydrolysates were then dried under N2, redissolved in H2O, sonicated for 7 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars, and for mannose-6-phosphate with a known number of moles was hydrolyzed in the same manner and at the same time as the sample. Four concentration of the neutral and amino sugar standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) and mannose-6-phosphate (640, 1280, 2560, 5120 picomoles per injection) were prepared to establish a calibration equation. The number of moles of each sugar in the sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars and mannose-6-phosphate 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 mannose-6-phosphate were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used eluents A, degassed nanopure water and B, 200 mM NaOH for neutral and amino sugars, and C, 100 mM NaOH and D, 1 M sodium acetate in 100 mM NaOH for mannose-6-phosphate. Injections were made every 40 minutes for neutral and amino sugar determination and every 35 minutes for mannose-6-phosphate determination. 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). Instrument control and data acquisition were accomplished using Dionex chromeleon software.