CHAIN MAPPING BY CTA-SAX AND SIZE EXCLUSION/ESI-MS OF 5 SAMPLES

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。 CTA SAX-HPLC法 样品用水溶解(20 mg/mL)，10°L进样到Phenomeex Gemini-NX C18色谱柱(3？m，250×4.6 mm)，涂有十六烷基三甲基硫酸氢铵。分析物通过232 nm处的紫外光吸收进行检测。使用以下系统通过盐梯度(表1)实现分离： 溶剂A：水(加入COCC调至pH 3.0)。甲磺酸)；溶剂B：2.5M甲磺酸，pH 2.5。 制备性凝胶过滤 采用TSKGel G2000SW色谱柱(7.8 mm ID×30 cm，5？m)，串联TSKGel保护柱(6.0 mm ID×4.0 cm，7？m)，以0.25M醋酸铵，pH 6.0为淋洗液，流速为0.5mL/min，进样量为25 mg/mLLMWH。紫外检测波长232 nm。含有四糖的部分(41.8-42.9min)从水中冷冻干燥3次，再用25°L水溶解，在同一柱子上重新纯化。将四糖组分从水中冷冻干燥3次。 电喷雾质谱学 质谱仪在Thermo Science LTQ Orbitrap Discovery仪器上直接输注，ESI模式下进行。进样速度：L/分钟，毛细管温度300C，毛细管电压-13V，喷雾电压-3.2KV，鞘气体流量18。

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. CTA SAX-HPLC The samples were dissolved in water (20 mg/mL) and 10 ¿L was injected onto a Phenomenex Gemini-NX C18 column (3 ¿m, 250x4.6 mm), coated with cetyltrimethylammonium hydrogen sulfate. Analytes were detected by their UV absorbance at 232 nm. Separation was effected by a salt gradient (Table 1) using the following system: Solvent A: Water (brought to pH 3.0 by addition of conc. methylsulfonic acid); Solvent B: 2.5 M methylsulfonic acid, pH 2.5. Preparative Gel Filtration Twenty-five ¿L of a 100 mg/mL aqueous solution of LMWH was injected onto two TSKGel G2000SW columns (7.8 mm ID X 30 cm, 5 ¿m), connected in series and equipped with a TSKGel guard column (6.0 mm ID X 4.0 cm, 7 ¿m), using 0.25 M ammonium acetate, pH 6.0 as eluent at a flow rate of 0.5 mL/min. Detection was by UV at 232 nm.. The fractions containing tetrasaccharide (41.8-42.9 min) were freeze-dried 3 times from water, redissolved in 25 ¿L water, and repurified on the same column. The tetrasaccharide containing fractions were freeze-dried 3 times from water. ESI Mass Spectrometry Mass spectrometry was performed on a Thermo Scientific LTQ Orbitrap Discovery instrument in ESI mode under direct infusion. Infusion rate was 5 ¿L/min, capillary temperature was 300 ¿C, capillary voltage -13 V, spray voltage -3.2 kV, and sheath gas flow rate was 18. Both the ion trap and the Orbitrap FT detector were used to acquire mass spectra.