MONOSACCHARIDE ANALYSIS OF O-LINKED OLIGOSACCHARIDES

O-连接低聚糖的单糖分析

基本信息

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
项目类别：
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

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。中心，不一定是研究者的机构。用丙酮:水清洗样品样品用丙酮:水(4:1)清洗两次，然后用100%丙酮清洗一次。将装有样品溶液的管置于冰中 15 分钟，4℃ 离心 15 分钟，除去将所得沉淀在真空干燥器中快速干燥。通过 ¿ 释放 O-连接聚糖-消除样品直接经受 ¿ -从糖蛋白上裂解O-连接聚糖的消除程序。简而言之，250 ¿将 L 50 mM NaOH 添加到样品中，然后检查 pH 值。是基本的，另外 250 ¿将含有 19 mg 硼氢化钠的 L 50 mM NaOH 添加到样品中，涡旋，并在 450°C 下孵育过夜，然后用 10% 乙酸中和并脱盐。通过Dowex树脂填充柱，并冻干。冻干后，清洁干燥的样品。在氮气流下用甲醇:乙酸 (9:1) 制备硼酸盐，释放出 O-连接寡糖。将样品通过 C18 sep pak 柱回收释放的 O-连接聚糖组分的等分部分。（提供~200克原始样品）被分配用于单糖成分分析。全O-甲基化碳水化合物的制备和纯化将样品中释放的 O-连接聚糖溶解在二甲基亚砜中，然后用 NaOH 甲基化，甲基碘（Ciucanu 和 Kerek，1984）通过添加水和全-O-甲基化来猝灭反应。用二氯甲烷提取碳水化合物，进一步清除污染物。简而言之，将聚糖装入 C18 sep pak 柱中，然后用纳米纯水洗涤。用 85% 乙腈洗脱纯化的聚糖，在氮气流下干燥并重新溶解。 MALDI-TOF 分析之前的甲醇。通过基质辅助激光解吸飞行时间质谱 (MALDI-TOF MS) 进行分析 MALDI-MS 使用 ¿ 在正离子模式下进行-二羟基苯甲酸（DHBA，20 mg/mL，50% 使用 4700 蛋白质组分析仪获得样品的全质谱。（应用生物系统公司）。 O-连接寡糖的单糖组成分析用于单糖成分分析的 O-连接聚糖等分试样用 400 ¿ 2.5 N 的 L 三氟乙酸 (TFA) 在 100¿ C下干燥4小时，将水解产物重悬于氮气流中。 H2O，在冰中超声处理 7 分钟，然后转移至注射瓶中。以相同的方式水解已知摩尔数的中性糖和氨基糖的标准品混合物并与样品同时注射四种浓度的标准混合物（每次注射 0.5、1.0、2.0 和 4.0 nmole）。准备建立校准方程，对样品中每种残留物的摩尔数进行定量。根据校准方程进行线性插值。使用配备 GP40 梯度泵的 Dionex DX500 系统通过 HPAEC 分析单糖， ED40 电化学检测器和包含不锈钢针的 Thermo-Separation AS3500 自动进样器。通过带有氨基捕集器的 Dionex CarboPac PA20 (3 x 150 mm) 分析柱分离糖。梯度程序使用洗脱液 A - 脱气纳米纯水、B - 200 mM NaOH 和 C - 100 mM NaOH 注射液。每 40 分钟进行一次。所有方法均基于 Hardy 和 Townsend (Hardy, M. R., 和 Townsend, R. R.，“糖蛋白衍生碳水化合物的高 pH 阴离子交换色谱”，1994 年，方法 Enzymol.230：208-225）使用 Dionex PeakNet 软件完成仪器控制和数据采集。版本 5.01。