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。 CTA SAX-HPLC 将样品溶于水（20 mg/mL）中，并将10 μ L进样至涂有十六烷基三甲基硫酸氢铵的Phenomenex Gemini-NX C18柱（3 μ m，250 × 4.6 mm）上。通过232 nm处的UV吸光度检测分析物。 使用以下系统通过盐梯度（表1）进行分离： 溶剂A：水（通过加入浓甲磺酸使pH值达到3.0）;溶剂B：2.5 M甲磺酸，pH值2.5。 澄清凝胶过滤 25磅将100 mg/mL LMWH水溶液注入两个TSKGel G2000 SW柱（7.8 mm ID X 30 cm，5 µ m），串联连接并配备TSKGel保护柱（6.0 mm ID X 4.0 cm，7 μ m），使用0.25 M乙酸铵（pH 6.0）作为洗脱剂，流速为0.5 mL/min。 将含有四糖的级分（41.8 - 42.9 min）从水中冷冻干燥3次，再溶解于25 μ L水中，并在同一柱上再纯化。 将含有四糖的级分从水中冷冻干燥3次。 esi质谱 在Thermo Scientific LTQ Orbitrap Discovery仪器上以ESI模式在直接输注下进行质谱分析。 输注速率为5 L/min，毛细管温度为300 ℃，毛细管电压为-13 V，喷雾电压为-3.2 kV，鞘气流速为18。 离子阱和Orbitrap FT检测器均用于获取质谱。

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.