N-LINKED OLIGOSACCHARIDE PROFILING BY HPAEC

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： N-连接葡聚糖的释放 冻干样品(样品1~7，~1 mg；样品A~0.4 mg，样品B~0.7 mg)用纳米水溶解，加入100 mM磷酸二氢钠缓冲液(pH 7.5)和1%十二烷基硫酸钠w/v/1M巯基乙醇，在100oC变性5min。冷却至室温后，加入冰冷的1M氯化钾，放置在冰上1.0小时。随后，将试管以4oC的最大速度旋转10分钟，以将十二烷基硫酸钠的钾盐制成颗粒。将每个样品的上清液转移到另一个干净的微离心管中，用PNGase F处理，并在37℃下孵育一夜。酶消化后，每个消化都通过C18Sep pak小柱，碳水化合物部分(包含N-连接的多糖)用5%的冰醋酸洗脱和冷冻干燥。 HPAEC-PAD法测定低聚糖图谱 每个样品的干燥N-连接低聚糖用纳米纯水[20g/L]溶解，转移到小瓶中注射。采用Dionex ICS3000系统、梯度泵、电化学检测器和自动进样器对低聚糖进行高效毛细管电泳法分析。低聚糖用Dionex CarboPac PA100(4×250 mm)分析柱和保护柱分离。流动相为160 mM氢氧化钠(A)和1M醋酸钠溶液(B)，流速为1ml/min。低聚糖的分离采用以下梯度程序：0min，A=100%和B=0；90min，A=80%和B=20%；91min，A=50%和B=50%；100min，A=50%和B=50%；101min，A=100%和B=0；以及110min，A=100%和B=0。自动进样器进样量为15L，仪器控制和数据采集采用Dionex chromeleon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.