PROFILING OF N-LINKED GLYCANS BY MALDI-TOF MS

通过 MALDI-TOF MS 分析 N 连接聚糖

• 批准号: 8170803
• 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/8170803
• 关键词: 1-Propanol; Acetic Acids; Acetonitriles; Acids; Aliquot; Buffers; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Dialysis procedure; Digestion; Dimethyl Sulfoxide; Enzymes; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Heating; Hour; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Membrane; Methanol; Methylation; Methylene Chloride; Monosaccharides; Nitrogen; Peptide Hydrolases; Peptide N-glycohydrolase F; Peptides; Polysaccharides; Propanols; Proteins; Proteomics; Reaction; Research; Research Personnel; Resources; Salts; Sampling; Sep-Pak C18; Series; Source; Stream; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; base; 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. The samples were dialyzed using a Tube-O-Dialyzer (4.0 kDa cut-off membrane; G BioSciences) against nanopure water at 4oC for about 18 hours to remove salts and other contaminants. Nanopure water was replaced three times during the entire dialysis period. Release of N-linked glycans After dialysis, an aliquot of each sample (to provide ~125 mg based on original sample information) was taken for monosaccharide composition analysis. The remainder was used for N-linked and O-linked glycans analysis by mass spectrometry. Briefly, the samples were 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 each sample and incubated at 37oC overnight. At the end of enzyme digestion, the tubes were heated at 100oC for 5 min to inactivate the trypsin. The tryptic digests were further cleaned of contaminants by passing through a C18 sep pak cartridge. Once loaded in the cartridge, the samples were cleaned with 5% acetic acid, and the glycopeptides/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 eluates were dried initially under a stream of nitrogen and then lyophilized. The dried tryptic digests were 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 samples were passed through a C18 sep pak cartridge and N-linked glycans fractions were eluted with 5% acetic acid. The O-linked glycopeptides/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 carbohydrate (N-linked glycans) fractions were dried by lyophilization, whereas the O-linked glycopeptides/peptides fractions were dried initially under a stream of nitrogen gas and eventually lyophilized. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans 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 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% 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。 使用 Tube-O-Dialyzer（4.0 kDa 截止膜；G BioSciences）在 4℃ 下对纳米纯水进行透析约 18 小时，以去除盐和其他污染物。在整个透析期间更换了 3 次纳米纯水。 N-连接聚糖的释放 透析后，取出每个样品的等分试样（根据原始样品信息提供约 125 mg）用于单糖成分分析，其余样品用于通过质谱法进行 N-连接和 O-连接聚糖分析。用蛋白酶缓冲液（0.1 M Tris-HCl，0.01 M CaCl2，pH 8.2）溶解，并在 100oC 下加热 5 分钟使其变性冷却至室温后，将胰蛋白酶添加到每个样品中并在37℃下孵育过夜。酶消化结束时，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% 异丙醇连续洗脱糖肽/肽。 ％乙酸、40％异丙醇的5％乙酸和100％异丙醇溶液被干燥。最初在氮气流下，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。 -连接聚糖级分用5%乙酸洗脱。O-连接糖肽/肽用20%异丙醇串联洗脱。 5%乙酸、40%异丙醇的5%乙酸和100%异丙醇溶液通过冻干干燥碳水化合物（N-连接聚糖）级分，而O-连接糖肽/肽级分最初在干燥条件下干燥。氮气流并最终冻干。 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，通过质谱分析进行结构表征（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。 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。