GLYCAN STRUCTURE ANALYSIS

聚糖结构分析

• 批准号: 7956054
• 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/7956054
• 关键词: 1-Propanol; Acetic Acids; Acetone; Acetonitriles; Acids; Analytical Biochemistry; Buffers; Carbohydrates; Computer Retrieval of Information on Scientific Projects Database; Digestion; Dimethyl Sulfoxide; Dryness; Endoglycosidase F; Enzymes; Funding; Glycopeptides; Grant; Heating; Hour; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylene Chloride; New England; Nitrogen; Peptide N-Glycosidase; Peptides; Phase; Polysaccharides; Precipitation; Preparation; Propanols; Proteomics; Reaction; Research; Research Personnel; Resources; Sampling; Sep-Pak C18; Series; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Stream; Structure; Technology; Trypsin; United States National Institutes of Health; Vacuum; Water; ammonium bicarbonate; 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. Acetone precipitation Cold acetone was added to the sample, which was then centrifuged at 4 oC for 15 min and supernatant was removed. Cold acetone was added to the sample again and re-centrifuged. The sample was dried down in the speed vacuum. Release of N-linked glycans from glycopeptide The dried sample was dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4), and denatured immediately by heating at 100 ¿C for 5min prior to trypsin digestion at 37 ¿C for overnight. After trypsin digestion, the sample was heated at 100 ¿C for 5 min to deactivate the enzyme. The sample was applied to a C18 sep-pak cartridge. Before elution of glycopeptides and peptides, the sample adsorbed in the C18 sep-pak cartridge was cleaned with 5% acetic acid. Peptides and glycopeptides then were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol. The propanol fraction was dried, and then sample was treated with a second and third enzyme, peptide N-glycosidase F (New England BioLabs) and peptide N-glycosidase A (Calbiochem) incubated at 37 ¿C for 20 hours to release the N-linked glycans. After enzymatic digestion, the sample was passed through a C18 reversed phase cartridge to separate the N-linked glycans from the peptides. The glycan fraction of the sample was eluted with 5% acetic acid and then lyophilized. Preparation of the per-O-methylated carbohydrates The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Analytical Biochemistry 203, 101-108 (1992)). The reaction was quenched by addition of water, and per-O-methylated carbohydrates were extracted with dichloromethane. The organic phase was concentrated to dryness and then the glycans were passed through a C18 Sep-Pak, eluted with 85 % acetonitrile, dried under a stream of nitrogen, and dissolved in methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) Profiling of N-linked glycans was performed initially using MALDI/TOF-MS on a 4700 Proteomics analyzer (Applied Biosystems). Permethylated glycans (~1 ¿L) were crystallized on a MALDI plate with 1 ¿L of 2, 3-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50 % methanol) as matrix. All spectra were acquired in the reflector positive ion mode and averaged the spectra of 50 laser shots.

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。 丙酮沉淀 将冷丙酮加入到样品中，然后在4℃下离心15分钟，并再次将冷丙酮加入到样品中并重新离心，将样品在高速真空中干燥。 从糖肽中释放 N-连接聚糖 将干燥的样品溶解在碳酸氢铵缓冲液（50 mM，pH 8.4）中，并立即在 100°C 下加热使其变性。 C 5 分钟，然后在 37 ¿ 下进行胰蛋白酶消化胰蛋白酶消化后，将样品在 100 °C 下加热过夜。 C 5 分钟以使酶失活。在洗脱糖肽和肽之前，用 5% 乙酸清洁 C18 sep-pak 柱中吸附的样品。依次用 20% 异丙醇的 5% 乙酸溶液、40% 异丙醇的 5% 乙酸溶液洗脱将丙醇部分干燥，然后用第二种和第三种酶处理样品，肽N-糖苷酶F（New England BioLabs）和肽N-糖苷酶A（Calbiochem）在37℃下孵育。 C 20 小时以释放 N-连接聚糖 酶消化后，将样品通过 C18 反相柱，将 N-连接聚糖与肽分离。用 5% 乙酸洗脱样品的聚糖部分。然后冻干。 全O-甲基化碳水化合物的制备 将冻干的碳水化合物级分溶解在二甲亚砜中，然后用NaOH和碘甲烷甲基化（Analytical Biochemistry 203, 101-108 (1992)）。通过添加水猝灭反应，并用二氯甲烷萃取全O-甲基化碳水化合物。将有机相浓缩至干，然后将聚糖通过 C18 Sep-Pak，洗脱用 85% 乙腈溶解，在氮气流下干燥，并溶解在甲醇中，然后进行质谱分析。 基质辅助激光解吸电离飞行时间质谱 (MALDI/TOF-MS) 最初使用 MALDI/TOF-MS 在 4700 蛋白质组分析仪 (Applied Biosystems) 上对 N-连接聚糖进行分析 (~)。 1 ¿L) 在具有 1 ¿ 的 MALDI 板上结晶L 2, 3-二羟基苯甲酸（DHBA，20 mg/mL 50% 甲醇溶液）作为基质。所有光谱均在反射器正离子模式下采集，并对 50 次激光照射的光谱进行平均。