PROFILING OF N-LINKED GLYCANS BY MALDI-TOF MS

通过 MALDI-TOF MS 分析 N 连接聚糖

• 批准号: 8170756
• 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/8170756
• 关键词: Acetic Acids; Acetone; Acetonitriles; Acids; Buffers; Carbohydrates; Chloroform; Computer Retrieval of Information on Scientific Projects Database; Dimethyl Sulfoxide; Enzymes; Excision; Funding; Gases; Glass; Glycopeptides; Grant; Heating; Hour; Ice; Incubated; Institution; Ions; Isopropanol; Lasers; Link; Lipids; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylation; Methylene Chloride; Nitrogen; Peptide N-glycohydrolase F; Peptides; Phosphate Buffer; Polysaccharides; Precipitation; 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; ammonium bicarbonate; methyl iodide; polypeptide; sodium phosphate; sugar

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. Lipid extraction and protein precipitation All samples were transferred from their original containers into screw-cap glass tubes. Lipids were extracted from the samples two times with chloroform:methanol:water (4:8:3). After lipid extraction, protein was precipitated with acetone:water (4:1) in ice with the concomitant removal of free sugars and contaminants. Release of N-linked glycans About 1.5 mg of each of the samples was weighed into a microcentrifuge tube and dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4). The samples were placed in a heating block at 100oC for 5 min to denature protein. After cooling to room temperature, the samples were treated with trypsin and chymotypsin and incubated at 37oC overnight. Each of the tryptic-chymotryptic digests was passed through a C18 sep pak cartridge, cleaned with 5% acetic acid, and glycopeptides/peptides were eluted subsequently in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid and 100% isopropanol. The eluates were dried initially under a stream of nitrogen gas and eventually lyophilized. The dried eluates were dissolved with sodium phosphate buffer, treated with PNGase F and incubated at 37oC for 18 hours to release N-linked glycans from the polypeptide chains. After incubation, the enzyme (PNGase F) digests were passed through C18 sep pak cartridges and N-linked glycans fractions were eluted with 5% acetic acid and lyophilized. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The PNGase-F released N- linked glycans from the three samples 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 by addition of 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。 对于中心来说，它不一定是研究者的机构。 脂质提取和蛋白质沉淀 将所有样品从其原始容器转移到螺旋盖玻璃管中，用氯仿：甲醇：水（4：8：3）从样品中提取脂质两次。脂质提取后，用丙酮：水（4）沉淀蛋白质。 :1) 在冰中同时去除游离糖和污染物。 N-连接聚糖的释放 将每个样品约 1.5 mg 称入微量离心管中，并溶解在碳酸氢铵缓冲液（50 mM，pH 8.4）中。冷却至室温后，将样品置于加热块中 100°C 5 分钟，使蛋白质变性。 ，将样品用胰蛋白酶和糜蛋白酶处理并在 37°C 下孵育过夜，将每种胰蛋白酶-糜蛋白酶消化物通过。 C18 sep pak柱，用5%乙酸清洗，随后用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇系列洗脱糖肽/肽。氮气流并最终冻干。 将干燥的洗脱液用磷酸钠缓冲液溶解，用 PNGase F 处理并在 37°C 下孵育 18 小时，以从多肽链中释放 N 连接聚糖。孵育后，将酶 (PNGase F) 消化物通过 C18 sep pak 柱并进行纯化。 N-连接聚糖级分用 5% 乙酸洗脱并冻干。 碳水化合物的全 O 甲基化和 C18 sep-pak 柱纯化 将三个样品中释放的 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。添加水和全O-甲基化碳水化合物，用二氯甲烷萃取全O-甲基化聚糖，并通过通过过滤器进一步纯化。 C18 sep pak 柱，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。