N-LINKED OLIGOSACCHARIDE PROFILING BY HPAEC

通过 HPAEC 进行 N-连接寡糖分析

• 批准号: 8363115
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363115
• 关键词: 2-Mercaptoethanol; Acetic Acids; Carbohydrates; Computer software; Digestion; Funding; Grant; Ice; Incubated; Injection of therapeutic agent; Link; Methods; National Center for Research Resources; Oligosaccharides; Peptide N-glycohydrolase F; Phase; Phosphate Buffer; Polysaccharides; Potassium; Potassium Chloride; Principal Investigator; Pump; Research; Research Infrastructure; Resources; Salts; Sampling; Sep-Pak C18; Sodium Acetate; Source; Speed; System; Technology; Temperature; Tube; United States National Institutes of Health; Vial device; Water; cost; data acquisition; detector; instrument; programs; sodium phosphate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Release of N-linked glycans The lyophilized samples (Samples 1 thru 7, ~1 mg; Sample A ~0.4 mg; and Sample B ~0.7 mg) were dissolved with nanopure water, added with 100 mM sodium phosphate buffer (pH 7.5) and 1% SDS w/v/1M ¿-mercaptoethanol and denatured at 100oC for 5 min. After cooling to room temperature, the mixture was added with ice-cold 1 M potassium chloride and placed on ice for 1.0 hr. Subsequently, the tubes were spun at 4oC, maximum speed for 10 min to pellet the potassium salts of SDS. The supernatant of each sample was transferred into another clean microcentrifuge tube, treated with PNGase F and incubated at 37oC overnight. After enzymatic digestion, each of the digests was passed through a C18 sep pak cartridge and the carbohydrate fraction (containing N-linked glycans) was eluted with 5% acetic acid and lyophilized. Oligosaccharide Profiling by HPAEC-PAD The dried N-linked oligosaccharides of each sample were dissolved with nanopure water [20 ¿g/¿L] and transferred into vials for injection. The oligosaccharides were analyzed by HPAEC using a Dionex ICS3000 system equipped with a gradient pump, an electrochemical detector, and an autosampler. The oligosaccharides were separated by a Dionex CarboPac PA100 (4 x 250 mm) analytical column with a guard column. The mobile phase eluents used were 160 mM NaOH (A) and 1 M sodium acetate in 160 mM NaOH (B) at a flow rate of 1 ml/min. Separation of oligosaccharides was accomplished with the following gradient program: 0 min, A=100% & B=0; 90 min, A=80% & B=20%; 91 min, A=50% & B=50%; 100 min, A=50% & B=50%; 101 min, A=100% & B=0; and 110 min, A=100% & B=0. Injection volume of the autosampler was set at 15 ¿L. Instrument control and data acquisition were accomplished using Dionex chromeleon software.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 方法： N-连接聚糖的释放 将冻干样品（样品 1 至 7，约 1 毫克；样品 A 约 0.4 毫克；样品 B 约 0.7 毫克）用纳米纯水溶解，添加 100 mM 磷酸钠缓冲液（pH 7.5）和 1% SDS w/v /1M ¿ -巯基乙醇并在 100℃ 下变性 5 分钟。冷却至室温后，向混合物中加入冰冷的 1 M 氯化钾，并置于冰上 1.0 小时。随后，将管在 4℃ 下以最大速度旋转 10 分钟。沉淀 SDS 的钾盐。将每个样品的上清液转移至另一个干净的微量离心管中，用 PNGase F 处理并在 30℃下孵育。 37°C 酶消化过夜后，将每种消化物通过 C18 sep pak 柱，用 5% 乙酸洗脱碳水化合物级分（含有 N-连接聚糖）并冻干。 通过 HPAEC-PAD 进行寡糖分析 将每个样品的干燥N-连接寡糖用纳米纯水溶解[20 ¿克/克L] 并转移到小瓶中进行注射，使用配备梯度泵、电化学检测器和自动进样器的 Dionex ICS3000 系统对寡糖进行分析。通过 Dionex CarboPac PA100 (4 x 250 mm) 分析仪对寡糖进行分离。使用的流动相洗脱液为 160 mM NaOH (A) 和 1 M 乙酸钠。 160 mM NaOH (B)，流速为 1 ml/min，通过以下梯度程序完成寡糖的分离：0 分钟，A=100% & B=0；90 分钟，A=80% & B=20。 %; 91 分钟，A=50% & B=50%；100 分钟，A=50% & B=50%； &B=0;和110分钟，A=100% &B=0，自动进样器的注射体积设置为15¿ L. 使用 Dionex chromeleon 软件完成仪器控制和数据采集。