STRUCTURAL ANALYSIS AND COMPOSITION ANALYSIS OF OLIGOSACCHARIDES

低聚糖的结构分析和成分分析

• 批准号: 8170761
• 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/8170761
• 关键词: Acetic Acids; Acetone; Acetonitriles; Acetylation; Acids; Amino Sugars; Analytical Biochemistry; Blood capillaries; Borates; Carbohydrates; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; Detection; Dimethyl Sulfoxide; Dryness; Electrons; Excision; Funding; Gases; Glycopeptides; Glycosides; Grant; Helium; Ice; Incubated; Institution; Ions; Lasers; Link; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Methanol; Methylene Chloride; Monosaccharides; Nitrogen; Oligosaccharides; Peptides; Phase; Plant Resins; Polysaccharides; Precipitation; Preparation; Procedures; Proteins; Proteomics; Reaction; Research; Research Personnel; Resources; Sampling; Scanning; Sep-Pak C18; Series; Silicon Dioxide; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Temperature; United States National Institutes of Health; Water; acetic anhydride; beta-Glucans; capillary; detector; ionization; methyl iodide; pyridine; sodium borohydride

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. Removal of contaminants by Acetone precipitation Acetone:water (4:1) was added to the samples (Als3 QCS #19186 and Als3 Pre-Q Absorber). The samples were placed on ice for 15 minutes and then centrifuged at 4 ¿C for 15 minutes to pellet the protein. The supernatant was removed. Cold acetone:water (4:1) was added to the samples again and re-centrifuged. Furthermore, the pellets were washed with 100% Acetone. Finally, the pellets were dried down under a nitrogen stream. ¿-Elimination, Desalting, Borate removal O-linked carbohydrates were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 250 ¿L of 50 mM NaOH were added to the samples (~100 ¿g) and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the sample and voltexed and incubated overnight at 45 ¿C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then were lyophilized. Dried sample was cleaned of borate with methanol: acetic acid (9:1) solution under a stream of nitrogen gas. The samples were then passed through a C18 reversed phase cartridge to separate the O-linked glycans from the peptides. The O-linked glycan fraction of the samples were eluted with 5% acetic acid and then lyophilized. Monosaccharide composition of O-linked glycans We prepared two sets of each samples to check evidence of beta-glucans in the samples. A set is O-linked carbohydrates were cleaved from the glycopeptides by ¿-elimination procedures without acetone precipitation and another set is with acetone precipitation. The monosaccharide composition of O-linked glycans of Als3 QCS #19186 and Als3 Pre-Q Absorber were analyzed by GC-MS. Methyl glycosides were prepared from a dried sample by methanolysis with 1 M HCl in methanol at 80¿C (18 h), followed by re-N-acetylation with pyridine and acetic anhydride in methanol for detection of amino sugars. The samples then were O-per-trimethylsilylated (TMS) with Tri-Sil (Pierce) at 80¿C. These procedures were carried out as described previously in Merkle and Poppe (1994); York, et al. (1985). GC/MS analysis of the TMS methyl glycosides was performed on a Hewlett Packard 5890 series  GC interfaced to a 5970 mass selective detector (MSD), electron impact ionization mode at 70 eV, and ions were scanned from 50 to 550 m/z, using a 0.25 mm ¿ 30 m fused silica capillary column (EC1, Alltech Associates, Deerfield, IL). The carrier gas was Helium. GC was started at 80¿C and held for 2 min and then increased temperature to 160¿C at a rate of 20¿C/min and held for 2 min; to 200¿C at a rate of 2¿C/min; to 250¿C at a rate of 5¿C/min. GC was held at the final temperature for 11 min. Preparation of the per-O-methylated carbohydrates The lyophilized carbohydrate fractions were 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 O- per-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 and O-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: water) as matrix. All spectra were acquired in the reflector positive ion mode and averaged 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。 通过丙酮沉淀去除污染物 将丙酮:水 (4:1) 添加到样品中（Als3 QCS #19186 和 Als3 Pre-Q Absorber） 将样品置于冰上 15 分钟，然后在 4 ℃ 下离心。 C 15 分钟以沉淀蛋白质。再次将冷丙酮:水(4:1)加入样品中并重新离心，最后用100%丙酮洗涤沉淀。在氮气流下干燥。 ¿ -消除、脱盐、去除硼酸盐 O-连接碳水化合物通过 ¿ 从糖肽上裂解下来- 简单地说，250 ¿将 L 50 mM NaOH 添加到样品中 (~100 µg)，然后检查 pH 值，确定 pH 值呈碱性后，再添加 250 µg。将 L 含有 19 mg 硼氢化钠的 50 mM NaOH 添加到样品中，涡旋并在 45°C 下孵育过夜。 C. 然后用 10% 乙酸中和孵育的样品，并通过 DOWEXTM 树脂填充柱（50W x 8 100，Sigma Aldrich）脱盐，然后用甲醇：乙酸（）冻干干燥样品中的硼酸盐。然后，在氮气流下将样品通过 C18 反相柱，将 O-连接聚糖与 9:1) 溶液分离。样品的 O-连接聚糖部分用 5% 乙酸洗脱，然后冻干。 O-连接聚糖的单糖组成 我们准备了两组样品来检查样品中 β-葡聚糖的证据，一组是 O-连接碳水化合物被 ¿ - 无需丙酮沉淀的消除程序，另一组使用丙酮沉淀的程序 通过 GC-MS 通过甲醇解法对 Als3 QCS #19186 和 Als3 Pre-Q Absorber 的 O-连接聚糖的单糖组成进行分析。 1 M HCl 甲醇溶液，80° C（18小时），然后用吡啶和乙酸酐在甲醇中重新N-乙酰化以检测氨基糖，然后用Tri-Sil（Pierce）在80°下对样品进行O-全三甲基硅烷化（TMS）。 C. 这些程序按照 Merkle 和 Poppe (1994) 中所述进行；在连接至 5970 质量的 Hewlett Packard 5890 系列 GC 上进行 TMS 甲基糖苷的 GC/MS 分析。选择性检测器 (MSD)，电子碰撞电离模式为 70 eV，离子扫描范围为 50 至 550 m/z，使用0.25 毫米 ¿ 30 m 熔融石英毛细管柱（EC1，Alltech Associates，Deerfield，IL） 气相色谱在 80° 下启动。 C并保持2分钟，然后将温度升高至160°C C 速率为 20¿ C/min 并保持 2 分钟至 200¿ C 的速率为 2¿ C/分钟；至250° C 的速率为 5¿ C/min. GC 在最终温度下保持 11 分钟。 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O-全甲基化碳水化合物。有机将相浓缩至干，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 连接和 O 连接聚糖进行分析。全甲基化聚糖 (~1 ¿L) 在具有 1 ¿L 的 MALDI 板上结晶L 2, 3-二羟基苯甲酸（DHBA，50% 甲醇：水中的 20 mg/mL 溶液）作为基质。所有光谱均在反射器正离子模式下采集，并获得 50 次激光照射的平均光谱。