MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 样品从原来的容器转移到螺帽管中，在氮气中干燥，用水重新溶解，将溶液平均分成两等份，然后冷冻干燥。一个等分用于中性和氨基糖分析，另一个等分用于唾液酸分析。中性糖和氨基糖等份在100℃下用2.0N三氟乙酸(TFA)400°L水解4h，唾液酸等分在80℃用2.0M醋酸400°L水解3h，所得水解物冷冻干燥，在水中悬浮，在冰中超声7min，转移到注射瓶中。 中性糖、氨基糖和唾液酸标准的混合物与已知摩尔数的标准以相同的方式同时进行水解。制备了4种浓度的标准混合物(每次注射0.5、1.0、2.0和4.0nmol.)以建立校正方程。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。中性糖和氨基糖分别用Dionex CarboPac PA20(3×150 mm)分析柱和氨基捕捉器分离。梯度程序使用洗脱剂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40分钟注射一次，唾液酸每35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. 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 transferred from the original container to a screw cap tube, dried under N2,, redissolved with H2O and divided equally the solution into two aliquots, and lyophilized. One aliquot was designated for neutral and amino sugars analysis with the other aliquot for sialic acids analysis. The aliquot intended for neutral and amino sugars was hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 h, whereas the aliquot for sialic acids was hydrolyzed with400 ¿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 sample. Four concentration of standard mix (0.5, 1.0, 2.0, and 4.0 nmoles per injection) 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 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 - 1M sodium acetate in 100 mM NaOH for sialic acids. Injections were made every 40 minutes for neutral and amino sugars and every 35 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).