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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 方法： 释放N连接的聚糖 将冻干的样品（样品1至〜1 mg；样品A〜0.4 mg；和样品B〜0.7 mg）用纳米水溶解，添加100 mm磷酸钠缓冲液（pH 7.5）和1％SDS W/V/V/V/1M。 -MercaptoEthanol，并在100oc中脱温，以100oc为5 min。冷却至室温后，将混合物与冰冷的1 M氯化钾一起添加，并将其放在冰上1.0小时。随后，管子以4oC旋转，最大速度10分钟，以颗粒颗粒的钾盐盐。将每个样品的上清液转移到另一个用PNGase F处理的清洁微输出管中，并在37oC中孵育过夜。酶促消化后，每种消化物都通过C18 Sep Pak弹药筒，并用5％的乙酸洗脱碳水化合物馏分（含有N连接的聚糖）并溶出。 通过HPAEC-PAD进行寡糖分析 将每个样品的干燥的N连接寡糖溶解在纳米水[20€g/¿l]中，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采样器的Dionex ICS3000系统通过HPAEC分析了寡糖。寡糖通过带有防护柱的Dionex Carbopac PA100（4 x 250 mm）分析柱分离。所使用的流动相脱位为160 mM NaOH（A）和1 M乙酸钠在160 mM NaOH（B）中，流速为1 ml/min。通过以下梯度程序完成了寡糖的分离：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％； 101分钟，a = 100％＆b = 0;和110分钟，a = 100％＆b = 0。将自动采样器的注入体积设置为15`L.仪器控制，并使用Dionex Chromeleon软件完成了数据采集。