N-LINKED GLYCANS PROFILING BY MALDI-TOF-TOF MS

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 将约3.0 mg的每种糖蛋白用0.1 M Tris-HCl溶解，并在100 ℃下加热5 min以使蛋白质变性。 冷却至室温后，向样品中加入胰蛋白酶和胰凝乳蛋白酶，并在37 ℃下孵育过夜。 在酶消化结束时，将样品在100 ° C下加热5分钟以使酶失活。 胰蛋白酶-胰凝乳蛋白酶消化酶通过C18分离包柱清除污染物。 一旦加载到柱中，两个样品中的每一个都用5%乙酸清洁，并且用5%乙酸中的20%异丙醇、5%乙酸中的40%异丙醇和100%异丙醇连续洗脱糖肽/肽。 首先在氮气流下干燥洗脱液，然后冻干。 将干燥的糖肽用50 mM NaPO 4缓冲液（pH约7.5）溶解，用PNGase F处理，并在37 ℃下孵育过夜以释放N-连接聚糖。 在第二次酶消化结束时，将样品通过C18 sep pak柱，用5%乙酸洗脱N-连接聚糖级分并冻干。 碳水化合物的全O-甲基化和C18 sep-pak柱的纯化 将从糖蛋白释放的N-连接聚糖的PNGase-F全甲基化，用于通过质谱法进行结构表征（Anumula和Taylor，1992）。 将干燥的洗脱液用二甲亚砜溶解，然后用NaOH和碘甲烷甲基化。 通过加入水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。 基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）分析寡糖 用甲醇溶解全甲基化N-连接聚糖，并用？-二羟基苯甲酸（DHBA，20 mg/mL，溶于50%甲醇：水）基质结晶。 使用AB SCIEX TOF/TOF 5800（Applied Biosystem MDS Analytical Technologies）通过MALDI-TOF-TOF-MS以正离子模式进行样品中存在的聚糖的分析。

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 About 3.0 mg of each glycoprotein was dissolved with 0.1 M Tris-HCl and heated at 100oC for 5 min to denature the protein. After cooling to room temperature, trypsin and chymotrypsin were added to the samples and incubated at 37oC overnight. At the end of enzyme digestion, the samples were heated at 100oC for 5 min to deactivate the enzymes. The tryptic-chymotryptic digests were cleaned of contaminants by passing through a C18 sep pak cartridge. Once loaded in the cartridge, each of the two samples was cleaned with 5% acetic acid and the glycopeptides/peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol. The eluates were dried initially under a stream of nitrogen and then lyophilized. The dried glycopeptides were dissolved with 50 mM NaPO4 buffer (pH~7.5), treated with PNGase F and incubated at 37oC overnight to release the N-linked glycans. At the end of the second enzyme digestion, the samples were passed through a C18 sep pak cartridge and N-linked glycans fraction was eluted with 5% acetic acid and lyophilized. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans from the glycoproteins were permethylated for structural characterization by mass spectrometry (Anumula and Taylor, 1992). The dried eluates were dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with methylene chloride. Oligosaccharide Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF TOF-MS) The permethylated N-linked glycans were dissolved with methanol and crystallized with ¿-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) matrix. Analysis of glycans present in the samples was performed in the positive ion mode by MALDI-TOF-TOF-MS using AB SCIEX TOF/TOF 5800 (Applied Biosystem MDS Analytical Technologies).