PROTON NMR OF 1 SAMPLE

1 个样品的质子核磁共振

• 批准号: 8361833
• 负责人: Parastoo Azadi
• 金额: $ 0.18万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361833
• 关键词: Acetates; Acetone; Aliquot; Blood capillaries; COSY; Chemicals; Deuterium; Dimethyl Sulfoxide; Electrons; Freeze Drying; Funding; Grant; Infusion procedures; Left; Mass Fragmentography; Measures; Methods; Methylation; NMR Spectroscopy; NOESY; National Center for Research Resources; Phase; Polymers; Principal Investigator; Protons; Relative (related person); Research; Research Infrastructure; Resources; Running; Sampling; Silicon Dioxide; Sodium Hydroxide; Solutions; Source; Spectrometry, Mass, Electrospray Ionization; Sugar Alcohols; TOCSY; Trifluoroacetic Acid; Tube; United States National Institutes of Health; Water; acetic anhydride; base; capillary; cost; detector; genetic linkage analysis; ionization; methyl iodide; pyridine; seal

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: NMR Spectroscopy The sample was deuterium exchanged by dissolving in D2O and lyophilization. The sample was dissolved in 80 ¿L D2O and placed in a 3 mm Shigemi NMR tube. 1-D Proton, TOCSY and NOESY NMR spectra, run with water presaturation, and gradient enhanced COSY and HSQC spectra were acquired on a Varian Inova-800 MHz spectrometer at 298 K (25 ¿C). Chemical shifts were measured relative to internal acetone (¿H=2.218 ppm, ¿C=33.00 ppm). Permethylation 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 was freeze-dried and suspended in about 200 ¿l of dimethyl sulfoxide. The sample was 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 40 minutes. The base was then added and left for 10 minutes and more methyl iodided was added and left for 40 minutes. The addition of more methyl iodide and NaOH base were to insure complete methylation of the polymer. Linkage analysis Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121¿C), reduced with NaBD4, and acetylated using acetic anhydride/pyridine. The resulting PMAAs were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD (mass selective detector, electron impact ionization mode); separation was performed on a 30 m EC-1 bonded phase fused silica capillary column. ESI Mass spectrometry The permethylated sample was analyzed by direct infusion in 50 % MeOH solution on a ThermoFisher LTQ. Experimental details are available upon request.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： NMR光谱 通过溶解在D2 O中并冻干来对样品进行氘交换。将样品溶解在80 μ L D2 O中，并置于3 mm Shigemi NMR管中。在Varian Inova-800 MHz光谱仪上在298 K（25 ℃）下获得1-D质子、TOCSY和NOESY NMR光谱，用水预饱和运行，以及梯度增强COSY和HSQC光谱。 测量相对于内部丙酮的化学位移（H=2.218 ppm，C=33.00 ppm）。 全甲基化 对于糖基连接分析，将样品全甲基化、解聚、还原和乙酰化;并且通过气相色谱-质谱法（GC-MS）分析所得的部分甲基化的糖醇乙酸酯（PMAA），如约克等人（1985）酶学方法（Methods Enzymol. 118：3-40。 首先，将一等份样品冷冻干燥并混悬于约200 µ l二甲基亚砜中。然后通过Ciukanu和Kerek（1984）Carbohydr的方法对样品进行全甲基化。Res. 131：209-217（用氢氧化钠和碘甲烷在无水DMSO中处理）。 将样品置于NaOH碱中10分钟，然后加入碘甲烷并放置40分钟。然后加入碱并静置10分钟，再加入碘甲烷并静置40分钟。加入更多的甲基碘和NaOH碱以确保聚合物的完全甲基化。 连锁分析 样品处理后，使用2 M三氟乙酸水解全甲基化材料（在密封管中于121 ℃下2 h），用NaBD 4还原，并使用乙酸酐/吡啶乙酰化。 在与5975 C MSD（质量选择性检测器，电子碰撞电离模式）连接的Agilent 7890 A GC上分析所得PMAA;在30 m EC-1键合相熔融石英毛细管柱上进行分离。 ESI质谱 通过在ThermoFisher LTQ上直接输注50% MeOH溶液来分析全甲基化样品。实验细节可根据要求提供。