N-GLYCAN PROFILING MALDI-MS

N-聚糖分析 MALDI-MS

• 批准号: 7956047
• 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: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956047
• 关键词: 1-Propanol; Acetic Acids; Acetone; Acetonitriles; Acids; Buffers; Carbohydrates; Centrifugation; Citrates; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Enzymes; Excision; Freeze Drying; Funding; Gases; Glycopeptides; Grant; Heating; Ice; Incubated; Institution; Ions; Isopropanol; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylation; Methylene Chloride; Nitrogen; Oligosaccharides; Peptide Hydrolases; Peptides; Polysaccharides; Propanols; Proteins; Proteomics; Reaction; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Series; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Technology; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; methyl iodide

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 samples were cleaned with acetone:water (4:1) two times and 100% acetone once. The preceding cleaning step was performed by placing the tubes of sample solutions in ice for 15 min, centrifugation at 4oC for 15 min and removal of supernatant. The resulting pellets were 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 Na citrate buffer (pH~4.5), treated with PNGase A 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 first with 5% acetic acid followed by the elution of O-glycopeptides/peptides fraction with 100% isopropanol. The carbohydrate (N-linked glycans) fraction was dried by lyophilization, whereas the isopropanol fraction was dried under a stream of nitrogen gas and stored. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The N- linked glycans were permethylated for oligosaccharide profiling (Ciucanu and Kerek, 1984). The dried eluate was 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 dichloromethane. Per- O-methylated glycans were cleaned of contaminants. Briefly, the glycans were dissolved in 1:1 methanol:water and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrates were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry. Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-TOF-MS was performed in the reflector positive ion mode using ¿-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectrum of the sample was obtained by using a 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 样品用丙酮:水(4:1)清洗两次，并用100%丙酮清洗一次。 将装有样品溶液的试管置于冰中 15 分钟，4℃ 下离心 15 分钟，然后取出 将所得沉淀在N 2 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> 将清洁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分钟使蛋白质变性后冷却至室温，将胰蛋白酶添加到样品中并在30℃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。 胰蛋白酶消化物装入后通过 C18 sep pak 柱进一步清除污染物。 纯化柱，用 5% 乙酸清洗样品，并用 5% 乙酸连续洗脱糖肽和肽。 20% 异丙醇的 5% 乙酸溶液、40% 异丙醇的 5% 乙酸溶液和 100% 异丙醇 将洗脱液干燥。 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 柠檬酸钠缓冲液 (pH~4.5) 溶解，用 PNGase A 处理并在 37oC 过夜以释放 N-连接聚糖 在第二次酶消化结束时，样品通过。 通过 C18 sep pak 柱，首先用 5% 乙酸洗脱 N-连接聚糖组分，然后用 用 100% 异丙醇洗脱 O-糖肽/肽级分。 通过冻干干燥，而异丙醇部分在氮气流下干燥并储存。 碳水化合物的全 O 甲基化和 C18 sep-pak 柱纯化 N-连接聚糖被全甲基化用于寡糖分析（Ciucanu 和 Kerek，1984）。 用二甲亚砜溶解，然后用NaOH和碘甲烷甲基化。通过NaOH猝灭反应。 添加水，并用二氯甲烷萃取全O-甲基化碳水化合物。 简而言之，将聚糖溶解在 1:1 甲醇:水中并加载到 C18 中。 sep pak 柱，然后用纳米纯水洗涤，用 15% 洗脱。 将乙腈装入螺旋盖管中，并将 85% 乙腈装入另一个螺旋盖管中，用 85% 乙腈洗脱聚糖。 乙腈在氮气流下干燥并用甲醇溶解以进行质量分析 光谱测定法。 通过基质辅助激光解吸飞行时间质谱 (MALDI-TOF MS) 进行分析 MALDI-TOF-MS 在反射器正离子模式下使用 ¿ -二羟基苯甲酸（DHBA，20 mg/mL，50% 使用 4700 蛋白质组分析仪获得样品的全质谱。 （应用生物系统公司）。