N-LINKED OLIGOSACCHARIDE PROFILING BY HPAEC

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： N-连接聚糖的释放 冻干样品（样品1至7，~1 mg;样品A ~0.4 mg;和样品B ~0.7 mg）用纳米纯水溶解，加入100 mM磷酸钠缓冲液（pH 7.5）和1% SDS w/v/1M？- 巯基乙醇，并在100 ℃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，在4 ℃下以最大速度旋转试管10分钟，以沉淀SDS的钾盐。 将每个样品的上清液转移到另一个干净的微量离心管中，用PNGase F处理，并在37 ℃下孵育过夜。 酶消化后，使每种酶通过C18 sep pak柱，用5%乙酸洗脱碳水化合物级分（含有N-连接的聚糖）并冻干。 通过HPAEC-PAD进行寡糖谱分析 将各样品的干燥N-连接寡糖用纳米纯水[20 μ g/μ L]溶解，并转移至小瓶中进行注射。 使用配备梯度泵、电化学检测器和自动进样器的Dionex ICS 3000系统，通过HPAEC分析寡糖。 通过带有保护柱的Dionex CarboPac PA 100（4 x 250 mm）分析柱分离寡糖。 用160 mM NaOH（A）和1 M乙酸钠的160 mM NaOH溶液（B）作为移动的流动相洗脱剂，流速为1 ml/min，用以下梯度程序完成寡糖的分离：0 min，A=100% & B=0; 90 min，A=80% & B=20%; 91 min，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。 自动进样器的进样体积设定为15 L。仪器控制和数据采集使用Dionex chromatography on软件完成。

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.