MONOSACCHARIDE ANALYSIS OF O-LINKED OLIGOSACCHARIDES

O-连接低聚糖的单糖分析

• 批准号: 7956051
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956051
• 关键词: Acetic Acids; Acetone; Acetonitriles; Acids; Aliquot; Amino Sugars; Anions; Borates; CD7 gene; Calibration; Carbohydrates; Centrifugation; Chromatography; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Computer software; Dimethyl Sulfoxide; Equation; Excision; Freeze Drying; Funding; Gases; Glycoproteins; Grant; Hydrolysis; Ice; Incubated; Injection of therapeutic agent; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Mole the mammal; Monosaccharides; Needles; Nitrogen; Oligosaccharides; Plant Resins; Polysaccharides; Preparation; Procedures; Proteomics; Protocols documentation; Pump; Reaction; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stainless Steel; Stream; System; Technology; Time; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Vacuum; Vial device; Water; base; data acquisition; detector; instrument; methyl iodide; programs; sodium borohydride; sugar

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. Sample cleaning by washing with acetone:water The sample was cleaned with acetone:water (4:1) two times and 100% acetone once. The preceding cleaning step was performed by placing the tube of sample solution in ice for 15 min, centrifugation at 4oC for 15 min and removal of supernatant. The resulting pellet was quick-dried in a vacuum dessicator. Release of O-linked glycans by ¿-elimination The sample was subjected directly to ¿-elimination procedures to cleave the O-linked glycans from the glycoprotein. Briefly, 250 ¿L of 50 mM NaOH were added to the sample and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the sample, vortexed, and incubated overnight at 450C. The incubated sample then was neutralized with 10% acetic acid, desalted by passing through a packed column of Dowex resins, and lyophilized. After lyophilization, the dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Released O-linked oligosaccharides were recovered by passing the sample through a C18 sep pak cartridge. An aliquot of the released O-linked glycans fraction (to provide ~200 ¿g of the original sample) was allocated for monosaccharide composition analysis. Preparation and purification of per-O-methylated carbohydrates The released O-linked glycans from the sample was dissolved in dimethyl sulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and redissolved with methanol prior to analysis by MALDI-TOF. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-MS was performed in the positive ion mode using ¿-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of the sample was obtained by using a 4700 Proteomics analyzer (Applied Biosystems). Monosaccharide composition analysis of O-Linked Oligosaccharides The O-linked glycan aliquot intended for monosaccharide composition analysis was hydrolyzed with 400 ¿L of 2.5 N trifluoroacetic acid (TFA) at 100¿C for 4 h. The hydrolysate was dried under a stream of nitrogen gas, resuspended in H2O, sonicated for 7 min in ice and transferred to an injection vial. 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 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 monosaccharides 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 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, B - 200 mM NaOH, and C - 100 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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 用丙酮清洗样品：水 用丙酮清洁样品：水（4：1）两次，一次100％丙酮。前面的清洁步骤是 通过将样品溶液的管放入冰中15分钟，在4oC下离心15分钟，然后去除 上清液。在真空dessicator中快速干燥所得的颗粒。 通过»释放O连接的聚糖�-灭绝 将样品直接进行``省略方法''，以从糖蛋白中清除O连接的聚糖。 简而言之，将250毫升的NaOH添加到样品中，然后检查pH。确定pH 是基本的，将含有19毫克硼氢化钠的50毫米NaOH的另外250°添加到样品中， 涡旋，并在450℃下孵育过夜。然后将孵育样品用10％乙酸中和，脱盐 通过穿过一列的脱沃克斯树脂柱，并冻干。冻干后，清洁干燥的样品 甲醇的硼酸盐：乙酸（9：1）在氮气流下。释放的O连接寡糖是 通过将样品通过C18 Sep Pak墨盒恢复。释放的O连锁聚糖分数的等分试样 （提供原始样品的〜200 g）进行单糖组成分析。 每甲基化碳液的制备和纯化 将样品中释放的O连接糖溶解在二甲基亚氧化二甲基亚氧化二甲基中，然后用NaOH甲基甲基甲基 甲基碘化物（Ciucanu和Kerek，1984）。通过加水和每甲基化来淬灭反应 用二氯甲烷提取碳水化合物。将 - 甲基化的甘氨酸进一步清洁污染物。 简而言之，将聚糖加载到C18 sep pak弹药筒中，然后用纳米水洗涤。然后是聚糖 用85％的乙腈洗脱。将纯化的聚糖干燥在氮气流下，并与 在MALDI-TOF进行分析之前，甲醇。 通过基质辅助激光解吸时间进行飞行时间质谱法（MALDI-TOF MS）进行分析 MALDI -MS使用 - 二羟基苯甲酸在正离子模式下进行（DHBA，20 mg/ml 50％ 甲醇：水）作为基质。通过使用4700蛋白质组学分析仪获得样品的全质量谱 （应用生物系统）。 O连锁寡糖的单糖组成分析 用于单糖组成分析的O连锁聚糖等分试样用400°L水解为2.5 n 在100°C处的三氟乙酸（TFA）4小时。在氮气流下将水解剂干燥，恢复 H2O，在冰中进行超声处理7分钟，并转移到注射小瓶中。 以相同的方式水解中性和氨基糖的标准和氨基糖的混合物。 并与样品同时。标准混合物的四个浓度（每注射0.5、1.0、2.0和4.0 nmoles） 准备建立校准方程。样品中每个住所的分子数量通过 校准方程式的线性插值。 使用配备GP40梯度泵的Dionex DX500系统通过HPAEC分析了单糖 ED40电化学探测器和一个含有不锈钢针的热分离AS3500自动采样器。 糖通过带有氨基陷阱的Dionex Carbopac PA20（3 x 150 mm）分析柱分离。这 梯度程序使用的脱位A-脱水纳米水，B -200 mm NaOH和C -100 mm NaOH。注射 每40分钟一次。所有方法均基于Hardy和Townsend所描述的协议（Hardy，M。R.，， 和汤森（R. R. 酶。 230：208-225）。使用Dionex Peaknet软件完成仪器控制和数据采集， 版本5.01。