PROFILING OF N-LINKED GLYCANS BY MALDI-TOF MS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 脂质提取和蛋白质沉淀 将所有样品从其原始容器中转移至螺旋盖玻璃管中。 用氯仿：甲醇：水（4：8：3）从样品中提取脂质两次。 脂质提取后，用丙酮：水（4：1）在冰中沉淀蛋白质，同时除去游离糖和污染物。 N-连接聚糖的释放 称取约1.5 mg各样品，置于微量离心管中，并溶于碳酸氢铵缓冲液（50 mM，pH 8.4）中。 将样品置于100 ℃的加热块中5分钟以使蛋白质变性。 冷却至室温后，用胰蛋白酶和糜蛋白酶处理样品，并在37 ℃下孵育过夜。 使每种胰蛋白酶-胰凝乳蛋白酶消化酶通过C18 sep pak柱，用5%乙酸清洗，随后用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。 洗脱液首先在氮气流下干燥，最后冻干。 用磷酸钠缓冲液溶解干燥的洗脱液，用PNGase F处理，并在37 ℃下孵育18小时，以从多肽链中释放N-连接聚糖。 孵育后，将酶（PNGase F）洗脱液通过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 对从三份样品中释放的PNGase-F N-连接聚糖进行全甲基化，以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。 通过C18 sep pak柱进一步纯化全-0-甲基化聚糖，用纳米纯水和15%乙腈洗涤。 最后，用85%乙腈洗脱清洁的全甲基化聚糖，并在氮气流下干燥。 基质辅助激光解吸飞行时间质谱（MALDI-TOF MS）分析 将干燥的纯化聚糖用甲醇溶解，并用？-二羟基苯甲酸（DHBA，20 mg/mL，溶于50%甲醇：水）基质结晶。 使用4700蛋白质组学分析仪（Applied Biosystems）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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lipid extraction and protein precipitation All samples were transferred from their original containers into screw-cap glass tubes. Lipids were extracted from the samples two times with chloroform:methanol:water (4:8:3). After lipid extraction, protein was precipitated with acetone:water (4:1) in ice with the concomitant removal of free sugars and contaminants. Release of N-linked glycans About 1.5 mg of each of the samples was weighed into a microcentrifuge tube and dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4). The samples were placed in a heating block at 100oC for 5 min to denature protein. After cooling to room temperature, the samples were treated with trypsin and chymotypsin and incubated at 37oC overnight. Each of the tryptic-chymotryptic digests was passed through a C18 sep pak cartridge, cleaned with 5% acetic acid, and glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The eluates were dried initially under a stream of nitrogen gas and eventually lyophilized. The dried eluates were dissolved with sodium phosphate buffer, treated with PNGase F and incubated at 37oC for 18 hours to release N-linked glycans from the polypeptide chains. After incubation, the enzyme (PNGase F) digests were passed through C18 sep pak cartridges and N-linked glycans fractions were 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 three samples 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. Per- O-methylated glycans were further purified by passing through a C18 sep pak cartridge, washed with nanopure water and 15% acetonitrile. Finally, cleaned permethylated glycans were eluted with 85% acetonitrile and dried under a stream of nitrogen gas. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) The dried purified 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 4700 Proteomics Analyzer (Applied Biosystems).