PROFILING OF N-LINKED GLYCANS BY MALDI-TOF MS

通过 MALDI-TOF MS 分析 N 连接聚糖

• 批准号: 8170757
• 负责人: Parastoo Azadi
• 金额: $ 0.13万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170757
• 关键词: Acetic Acids; Acetone; Acetonitriles; Acids; Buffers; Carbohydrates; Chloroform; Computer Retrieval of Information on Scientific Projects Database; Dimethyl Sulfoxide; Enzymes; Excision; Funding; Gases; Glass; Glycopeptides; Grant; Heating; Hour; Ice; Incubated; Institution; Ions; Isopropanol; Lasers; Link; Lipids; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylation; Methylene Chloride; Nitrogen; Peptide N-glycohydrolase F; Peptides; Phosphate Buffer; Polysaccharides; Precipitation; Proteins; Proteomics; Reaction; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Series; Source; Stream; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; ammonium bicarbonate; methyl iodide; polypeptide; sodium phosphate; sugar

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 seven 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).

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 脂质提取和蛋白质沉淀 将所有样品从原始容器转移到螺杆玻璃管中。用氯仿两次从样品中提取脂质：甲烷：水（4：8：3）。脂质提取后，用丙酮沉淀蛋白质：在冰中的水（4：1），并随同去除游离糖和污染物。 释放N连接的聚糖 将约1.5 mg的每个样品称重为微量离心管，并溶于碳酸氢铵缓冲液（50 mm，pH 8.4）。将样品在100oC的加热块中放置5分钟，以取代蛋白质。冷却至室温后，将样品用胰蛋白酶和辣椒蛋白处理，并在37oC中孵育过夜。每种胰蛋白酶 - 染色体杀手蛋白均通过C18 SEP PAK墨盒，用5％的乙酸清洁，然后在5％乙酸中以20％的异丙醇为单位，将糖肽/肽在5％的5％异丙醇中以5％的5％乙酸和100％的丙型丙二醇为生，然后在5％的5％乙酸中洗脱糖肽/肽。催珠最初在氮气流下干燥，并最终冻干。 将干燥的洗脱液用磷酸钠缓冲液溶解，用PNGase F处理，并在37oC下孵育18小时，以从多肽链中释放出N连接的聚糖。孵育后，将酶（PNGASE F）消化通过C18 Sep Pak弹药，并用5％乙酸洗脱N连接的聚糖级分，并冻干。 C18 Sep-pak墨盒的碳水合物的每甲基化和纯化 从七个样品中释放出N-连接的聚糖的PNGASE-F被氯化，以通过质谱法进行结构表征（Anumula and Taylor，1992）。将干燥的洗脱液与二甲基硫氧化物溶解，然后用NaOH和碘化甲基甲基化。通过添加水和每甲基化的碳含水液用甲基氯化物来淬灭反应。每甲基化的聚糖，通过穿过C18 Sep Pak墨盒，用纳米水洗涤和15％乙腈，进一步纯化。最后，用85％的乙腈洗脱清洁的苄氨基甲基化聚糖，并在氮气流下干燥。 通过基质辅助激光解吸时间进行飞行时间质谱法（MALDI-TOF MS）进行分析 将干燥的纯化糖溶解在甲醇中溶解，并用�-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（Applied Biosystems），通过MALDI-TOF-MS在阳性离子模式下对样品中提出的聚糖进行分析。