STRUCTURAL ANALYSIS OF O-LINKED OLIGOSACCHARIDES, MONOSACCHARIDE COMPOSITION

O-连接低聚糖、单糖组合物的结构分析

• 批准号: 7956050
• 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/7956050
• 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 An aliquot (~400 ¿g) 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 ~100 ¿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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中央赠款提供的资源 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，在列出的机构中可能会被压制 对于中心，这不一定是调查员的工厂。 用丙酮清洗样品：水 用丙酮清洁了等分试样（〜400»）：水（4：1）两次，100％的丙酮通过将PLE溶液放置在冰中15分钟，在中央部门进行100％的清洁步骤。 4oC持续15分钟，去除上清液。 通过»释放O连接的聚糖 - 淘汰 样品直接受到 - 脱离糖蛋白的O连接糖的脱离程序。 l将50 mm的NaOH添加到样品中，并在确定pH值时进行检查。 l加入50毫米NaOH的NaOH碳水化合物，涡旋，涡流，并在450°C中孵育过夜。在氮气流中用甲醇（9：1）清洗干燥的样品，通过将样品通过C18 Sep Pak弹药筒恢复。原始样品的g分配了单糖组成分析。 甲基化碳水化合物的制备和纯化 通过添加水和氧化二甲烷的O-甲基化碳水化合物的释放的O型糖。 C18 sep partridge用85％的乙腈洗脱了Glycans。 通过矩阵辅助激光测定飞行时间质谱（MALDI-TOF MS）进行分析 使用„在正离子模式下进行MALDI-MS进行 - 二羟基苯甲酸（DHBA，20 mg/ml，50％metrix。Zer（Applied Biosystems）。 O连锁寡糖的单糖组成分析。 用于单糖组成分析的O连锁的聚糖等分试样用400€液化液化。 1 100 n的2.5 n truoroaceticac（TFA） C持续4 h。 在标准混合物（0.5、1.0、2.0和4.0 Nmoles的样品中），将中性糖和氨基糖的标准品与已知数量的摩尔糖的混合在一起。样品中每个残基的摩尔数量通过校准来量化。 使用HPAEC分析了单糖，使用配备GP40梯度泵O-分离的DX500系统AS3500 AutoSampler conta intaine intainex carbopac PA20（3 x 150 mmmmmmmmmintical柱）每40分钟的纳米水de计划。使用Dionex Peaknet软件（版本5.01）完成了208-225的酶。