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 用丙酮清洁样品：水（4：1）两次，一次100％丙酮。前面的清洁步骤 通过将样品溶液的管放入冰中15分钟，在4oC下离心15分钟，然后去除 上清液。在N2的流中将产生的颗粒干燥。 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分钟以结染蛋白质。冷却至室温后，将胰蛋白酶添加到样品中并在 37oc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。一旦加载 用5％乙酸清洗样品，糖肽和胡椒粉串联洗脱 5％乙酸中的20％ISO丙醇，5％乙酸中的40％ISO丙醇和100％ISO丙醇。洗脱石干了 最初是在氮流下，然后冻干。 将干燥的胰蛋白酶消化物与50 mm Na柠檬酸盐缓冲液（pH〜4.5）溶解，并用PNGase A处理，并在 37oc过夜以释放N连接的聚糖。在第二酶消化的结束时，通过样品通过 首先，通过C18 SEP PAK弹药筒和N连接的聚糖馏分以5％的乙酸洗脱，然后是 用100％异丙醇的O-糖肽/肽分数洗脱。碳水合物（N连接的聚糖）是 通过冻干干燥，而异丙醇馏分在氮气流下干燥并储存。 C18 Sep-pak墨盒的碳水合物的每甲基化和纯化 N连接的甘氨酸被氯二苄酯进行寡糖分析（Ciucanu and Kerek，1984）。干洗车是 用二甲基硫氧化物溶解，然后用NaOH和碘化甲基甲基化。反应被淬灭 用二氯甲胺提取水和每甲基化的碳氢化物。每甲基化 将聚糖清洗为污染物。简而言之，将聚糖溶解在1：1甲醇：水中并加载到C18中 Sep Pak弹药筒，然后用纳米水洗涤。用15％洗脱每欧甲基碳水 乙腈进入螺杆管中，并在另一个螺钉管中用85％的乙腈进入。聚糖以85％洗脱 将乙腈在氮气流下干燥，并用甲醇溶解以通过质量分析 光谱法。 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 使用 - 二羟基苯甲酸（DHBA，20 mg/ml 50％），在反射剂正离子模式下进行MALDI-TOF-MS进行 甲醇：水）作为基质。通过使用4700蛋白质组学分析仪获得样品的全质量谱 （应用生物系统）。