MONOSACCHARIDE COMPOSITION ANALYSIS OF RELEASED N-LINKED GLYCANS 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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 研究方法： 将用于中性糖和氨基糖的释放的N-连接聚糖等分试样用400 <$L 2.0 N三氟乙酸（TFA）在100 <$C下水解4 h。 将水解产物冻干，重悬于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（每次进样0.5、1.0、2.0和4.0纳摩尔），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。 通过HPAEC使用Dionex DX 500系统分析中性糖和氨基糖，Dionex DX 500系统配备有GP 40梯度泵、ED 40电化学检测器和含有不锈钢针的Thermo-Separation AS 3500自动进样器。 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。 梯度程序使用洗脱液A -脱气的纳米纯水，和B - 200 mM NaOH。 每40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）。使用Dionex PeakNet软件（版本5.01）完成仪器控制和数据采集。

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.