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。 通过丙酮沉淀去除污染物 丙酮：将水（4：1）添加到样品中（ALS3 QCS＃19186和ALS3 Pre-Q吸收器）。将样品放在冰上15分钟，然后在4°C下离心15分钟，以颗粒。上清液被删除。冷丙酮：再次将水（4：1）添加到样品中并重新融合。此外，用100％丙酮洗涤颗粒。最后，将颗粒干燥在氮流下。 - 淘汰，脱盐，去除硼酸盐 O连接的碳水化合物通过``省略碳肽''糖肽裂解。简而言之，将250毫升的NaOH添加到样品（〜100»g）中，然后检查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.干燥样品用甲醇清洁硼酸盐：乙酸（9：1）溶液在氮气流下。然后，样品通过C18反向相墨盒，将O连接的聚糖与肽分开。将样品的O连接聚糖分数用5％乙酸洗脱，然后冻干。 O连锁聚糖的单糖组成 我们准备了两套样品，以检查样品中β-葡聚糖的证据。一组是O连接的碳水化合物，通过没有丙酮沉淀的渗漏程序从糖肽中裂解，另一组是丙酮沉淀。通过GC-MS分析了ALS3 QCS＃19186和ALS3 PRE-Q吸收仪的O连接聚糖的单糖组成。通过在80 ch（18小时）的甲醇中用1 M HCl的甲硅溶制备甲基糖苷，然后在甲基甲基甲基甲基和乙醇中重新甲基化，然后在甲基醇中重新乙酰化，以检测氨基糖。然后，样品是在80¿C处的三甲基二甲基硅烷基硅烷基硅烷基硅烷基硅烷基硅烷基硅烷基硅烷二烯丙基（TMS）。这些程序是如前所述在Merkle和Poppe（1994）中所述进行的；约克等。 （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）。载气是氦气。 GC从80°C开始，持续2分钟，然后以20°C/min的速度升高到160°C，并保持2分钟；到200 c，速率为2 c/min；以5 c/min的速率到250°C。 GC在最终温度保持11分钟。 每甲基化碳液的制备 将冻干的碳水化合物馏分溶于二甲基硫氧化物，然后用NaOH和甲基碘化甲基甲基甲基（分析生物化学203，101-108（1992）（1992年）。通过加入水，将氧化碳水化合物添加到二氯甲烷中，并通过甲基化的碳素含有二氯甲烷。 C18 SEP-PAK用85％的乙腈洗脱，在氮气流下干燥，并溶解在甲醇中，然后通过质谱分析。 最初，使用MALDI/TOF-MS在4700蛋白质组学分析仪（Applied Biosystems）上使用MALDI/TOF-MS对N-C-连接和O连接的聚糖进行基质辅助激光解电离电离电离质谱（MALDI/TOF-MS）分析。将苄苄化聚糖（〜1»l）在MALDI板上结晶，1°L的2、3-二羟基苯甲酸（DHBA，20 mg/ml溶液在50％甲醇：水：水）中为矩阵。所有光谱均在反射器正离子模式下获得，平均光谱为50激光镜头。