PROFILING OF N-LINKED GLYCANS BY MALDI-TOF MS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脂肪提取和蛋白质沉淀 所有样品都从原来的容器中转移到螺帽玻璃管中。样品用氯仿：甲醇：水(4：8：3)提取2次。提取脂肪后，用丙酮：水(4：1)在冰中沉淀蛋白质，同时去除游离糖和污染物。 N-连接葡聚糖的释放 每个样品重约1.5毫克，放入微量离心管中，并溶解在碳酸氢铵缓冲液(50 mm，pH 8.4)中。将样品放入100oC的加热块中5min变性蛋白质。冷却至室温后，用胰酶和凝乳酶处理样品，并在37℃下孵育一夜。每一个胰酶-胰凝乳酶消化液都通过C18Sep pak小柱，用5%的冰醋酸洗脱，然后用20%异丙醇5%冰醋酸、40%异丙醇5%冰醋酸和100%异丙醇串联洗脱。洗脱物最初在氮气气流下干燥，最后冷冻干燥。 干燥的洗脱液用磷酸二氢钠缓冲液溶解，用PNGase F处理，在37oC孵育18h，从多肽链上释放N-连接的多糖。孵育后，酶(PNGase F)消化通过C18Sep pak柱，N-连接的多聚糖组分用5%的冰醋酸洗脱并冷冻干燥。 碳水化合物的Per-O-甲基化及C18Sep-Pak柱纯化 从三个样品中释放的PNGase-F被全甲基化以用于质谱学结构表征(Anuula和Taylor，1992)。干洗脱液用二甲基亚砜溶解，然后用氢氧化钠和碘甲烷甲基化。加水使反应熄灭，用二氯甲烷提取全O-甲基化碳水化合物。通过C18Sep pak柱，用纳米纯水和15%乙腈洗涤，进一步纯化了Per-O-甲基化聚糖。最后，清洗过的过甲基化多糖用85%的乙腈洗脱，并在氮气气流下干燥。 基质辅助激光解吸飞行时间质谱仪(MALDI-TOF MS)分析 将干燥的纯化多糖用甲醇溶解，用二羟基苯甲酸(DHBA，20 mg/mL，50%甲醇：水)基质结晶。使用4700蛋白质组学分析仪(应用生物系统)，通过MALDI-TOF-TOF-MS以正离子模式对样品中存在的葡聚糖进行分析。

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).