MONOSACCHARIDE COMPOSITION ANALYSIS OF RELEASED N-LINKED GLYCANS 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。 方法： 将释放出来的中性糖和氨基糖用2.0N三氟乙酸(TFA)的400°L在100℃下水解4h，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。 以与样品相同的方式同时对已知摩尔数的中性糖和氨基糖的标准混合物进行水解。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。 中性糖和氨基糖的分析采用Dionex DX500系统，该系统配备了GP40梯度泵、ED40电化学检测器和带有不锈钢针的热分离AS3500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 NaOH。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)。仪器控制和数据采集使用Dionex PeakNet软件5.01版完成。

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. Methods: The released N-linked glycan aliquots intended for neutral and amino sugars were hydrolyzed with 400 ¿L of 2.0 N trifluoroacetic acid (TFA) at 100¿C for 4 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 with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. 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 were analyzed by HPAEC using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. 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. Injections were made every 40 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). Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.