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).

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 中心，不一定是研究者的机构。 用丙酮:水清洗样品 用冷丙酮:水 (4:1) 和 100% 冷丙酮清洗样品两次，并将所得颗粒在氮气流下干燥。 通过 MALDI-TOF MS 进行 N 连接寡糖分析 N-连接聚糖的释放 将清洗后的样品用蛋白酶缓冲液（0.1 M Tris-HCl、0.01 M CaCl2，pH 8.2）溶解，并在 100℃ 下加热 5 分钟，使蛋白质变性。冷却至室温后，向样品中加入胰蛋白酶，并在 3 ℃下孵育。 37℃过夜，酶消化结束后，将管在100℃下加热5分钟以灭活胰蛋白酶。 胰蛋白酶消化物通过 C18 sep pak 柱进一步清除污染物。一旦装入柱，用 5% 乙酸清洗样品，并用 5% 的 20% 异丙醇连续洗脱糖肽和肽。乙酸、40% 异丙醇的 5% 乙酸和 100% 异丙醇溶液 首先干燥洗脱液。在氮气流下，然后冻干。 将干燥的胰蛋白酶消化物用 50 mM 磷酸钠溶解，用 PNGase F 处理并在 37°C 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 柱和 N。用5％乙酸洗脱N-连接聚糖级分，通过冻干干燥碳水化合物(N-连接聚糖)级分。 碳水化合物的全 O 甲基化和 C18 sep-pak 柱纯化 将 PNGase-F 释放的 N 连接聚糖进行全甲基化，通过质谱进行结构表征（Anumula 和 Taylor，1992）。将干燥的洗脱液用二甲亚砜溶解，然后用 NaOH 和碘甲烷进行甲基化。用水和全碘猝灭反应。用二氯甲烷提取 O-甲基化碳水化合物，并通过 C18 sep pak 柱进一步纯化。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。 通过基质辅助激光解吸飞行时间质谱 (MALDI-TOF MS) 进行分析 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 使用 4700 蛋白质组分析仪（Applied Biosystems）通过 MALDI-TOF-TOF-MS 以正离子模式对样品中存在的聚糖进行分析。