N-GLYCAN PROFILING BY MALDI-MS

通过 MALDI-MS 进行 N-聚糖分析

• 批准号: 8170766
• 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/8170766
• 关键词: 1-Propanol; Acetic Acids; Acetone; Acetonitriles; Acids; Buffers; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Enzymes; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Heating; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylation; Methylene Chloride; Nitrogen; Oligosaccharides; Peptide Hydrolases; Peptide N-glycohydrolase F; Peptides; Polysaccharides; Propanols; 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; methyl iodide; sodium phosphate

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. Sample cleaning by washing with acetone:water The sample was cleaned with cold acetone:water (4:1) and 100 % cold acetone two times and the resulting pellet was dried under a stream of N2. N-linked oligosaccharide profiling by MALDI-TOF MS Release of N-linked glycans The cleaned sample was dissolved with protease buffer (0.1 M Tris-HCl, 0.01 M CaCl2, pH 8.2), and heated at 100oC for 5 min to denature the protein. After cooling to room temperature, trypsin was added to the sample and incubated at 37oC overnight. At the end of enzyme digestion, the tube was heated at 100oC for 5 min to inactivate the trypsin. The tryptic digest was further cleaned of contaminants by passing through a C18 sep pak cartridge. Once loaded in the cartridge, the sample was cleaned with 5% acetic acid and the glycopeptides and 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 eluate was dried initially under a stream of nitrogen and then lyophilized. The dried tryptic digest was dissolved with 50 mM sodium phosphate, treated with PNGase F and incubated at 37oC overnight to release the N-linked glycans. At the end of the second enzyme digestion, the sample was passed through a C18 sep pak cartridge and N-linked glycans fraction was eluted with 5% acetic acid. The carbohydrate (N-linked glycans) fraction was dried by lyophilization. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans was permethylated for structural characterization by mass spectrometry (Anumula and Taylor, 1992). The dried eluate was dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched with 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。 用丙酮清洗样品：水 用冷丙酮清洁样品：水（4：1）和100％冷丙酮两次，并在N2流下干燥所得的颗粒。 MALDI-TOF MS的N连接寡糖分析 释放N连接的聚糖 将清洁的样品用蛋白质缓冲液（0.1 M Tris-HCl，0.01 M CaCl2，pH 8.2）溶解，并在100oC中加热5分钟以使蛋白质变性。冷却至室温后，将胰蛋白酶添加到样品中并在37oC中孵育过夜。在酶消化的结尾，将管子在100oC中加热5分钟，以使胰蛋白酶失活。 通过穿过C18 Sep Pak弹药筒，胰蛋白酶的文摘进一步清洁了污染物。一旦装入墨盒，将样品用5％乙酸清洗，糖肽和辣椒在5％乙酸中用20％ISO丙醇串联洗脱，在5％的5％乙酸中40％ISO丙醇和100％ISO丙醇。洗脱液最初是在氮流下干燥的，然后冻干。 将干燥的胰蛋白酶消化物与50 mM磷酸钠溶解，并用PNGase F处理，并在37oC中孵育过夜以释放N连接的聚糖。在第二个酶消化的结束时，样品通过C18 sep pak弹药筒，并用5％乙酸洗脱N连接的聚糖馏分。碳水化合物（N连接的聚糖）的部分是通过冻干干燥的。 C18 Sep-pak墨盒的碳水合物的每甲基化和纯化 PNGASE-F释放的N连接的聚糖被氯化，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。用水淬灭反应，并用甲基氯化物提取每种甲基化的碳水合物。通过穿过C18 Sep Pak弹药筒，用纳米水洗涤和15％乙腈，进一步纯化了o-甲基化的聚糖。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。