GLYCOSYL COMPOSITION, LINKAGE AND MALDI MS ANALYSIS

糖基组成、连接和 MALDI MS 分析

• 批准号: 8170792
• 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/8170792
• 关键词: Acetates; Acetonitriles; Acetylation; Aliquot; Amino Sugars; Blood capillaries; Client; Computer Retrieval of Information on Scientific Projects Database; Detection; Dialysis procedure; Dimethyl Sulfoxide; Electrons; Esters; Funding; Glycosides; Grant; Hour; Hydrolysis; Institution; Left; Mass Fragmentography; Methanol; Methods; Methylation; Monosaccharides; Oligosaccharides; Phase; Polymers; Procedures; Research; Research Personnel; Resources; Running; Sampling; Silicon Dioxide; Sodium Hydroxide; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Sugar Alcohols; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Uronic Acids; Water; acetic anhydride; base; capillary; detector; genetic linkage analysis; ionization; mass spectrometer; methyl iodide; pyridine; seal

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. Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. Methyl glycosides were first prepared from dry sample provided by the client by methanolysis in 1 M HCl in methanol at 80¿C (18-22 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80¿C (0.5 hours). [These procedures were carried out as previously described in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15; York, et al. (1985) Methods Enzymol. 118:3-40.] GC/MS analysis of the TMS methyl glycosides was performed on an HP 6890 GC interfaced to a 5975b MSD, using a All Tech EC-1 fused silica capillary column (30m ¿ 0.25 mm ID). For glycosyl linkage analysis, the sample was permethylated, depolymerized, reduced, and acetylated; and the resultant partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Initially, an aliquot of the sample after dialysis was suspended in about 200 ul of dimethyl sulfoxide. The samples were then permethylated by the method of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The sample was subjected to the NaOH base for 10 minutes then methyl iodide was added and left for 20 minutes. The base was then added for 10 minutes and finally more methyl iodided was added for 20 minutes. This addition of more methyl iodide and NaOH base was to insure complete methylation of the polymer. Following sample workup, the permethylated material was reduced by superdeuteride to reduce methyl ester of uronic acid, and then hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121¿C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode); separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. MALDI MS The sample was dissolved in deionized water (1mg/ml) and 1 ul of the solution was spotted on a spot of DHB dried from acetonitrile/water (1:1), and subjected to MALDI MS on a Bruker MicroFlex Mass Spectrometer which was run in the positive mode. All masses were calibrated by malto-oligosaccharide controls run immediately before the samples.

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。 通过气相色谱/质谱联用 (GC/MS) 对通过酸性甲醇分解样品产生的单糖甲基糖苷的全 O-三甲基甲硅烷基 (TMS) 衍生物进行糖基组成分析。 首先通过客户提供的干燥样品在 1 M HCl 的甲醇溶液中于 80° 下进行甲醇分解来制备甲基糖苷。 C（18-22小时），然后用甲醇中的吡啶和乙酸酐进行再N-乙酰化（用于检测氨基糖），然后通过在80℃下用Tri-Sil（Pierce）处理来对样品进行全O-三甲基甲硅烷基化。 ¿ C（0.5小时）[这些程序按照Merkle和Poppe（1994）Methods Enzymol.230：1-15；York等人（1985）Methods Enzymol.118：3-40中所述进行。 TMS 甲基糖苷的 /MS 分析是在连接 5975b MSD 的 HP 6890 GC 上进行的，使用 All Tech EC-1 熔融石英毛细管柱（30m ¿ 0.25 mm ID）。 对于糖基键分析，样品被全甲基化、解聚、还原和乙酰化；并且按照 York 等人 (1985)Methods Enzymol 的描述，通过气相色谱-质谱 (GC-MS) 分析所得部分甲基化糖醇乙酸酯 (PMAA)。 118:3-40。 最初，将透析后的等分样品悬浮于约200ul二甲基亚砜中，然后通过Ciukanu和Kerek(1984)CarboHydr.131:209-217的方法进行全甲基化(用氢氧化钠和甲基化处理)。将样品置于 NaOH 碱中 10 分钟，然后添加甲基碘并静置 10 分钟。然后在10分钟内添加碱，最后在20分钟内添加更多的碘甲烷和NaOH碱以确保在样品处理后，全甲基化材料被减少。超氘化物还原糖醛酸甲酯，然后用2 M三氟乙酸水解（121℃密封管中2小时），还原使用 NaBD4 进行乙酰化，并使用乙酸酐/三氟乙酸在连接至 5970 MSD（质量选择检测器，电子轰击电离模式）的 Hewlett Packard 5890 GC 上对所得 PMAA 进行分析，并在 30 m Supelco 2330 键合相上进行分离。熔融石英毛细管柱。 马尔迪质谱 将样品溶解在去离子水 (1mg/ml) 中，并将 1ul 溶液点在乙腈/水 (1:1) 干燥的 DHB 点上，并在运行的 Bruker MicroFlex 质谱仪上进行 MALDI MS在阳性模式下，所有质量均通过在样品之前立即运行的麦芽低聚糖对照进行校准。