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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 糖基组成分析进行了组合的气相色谱/质谱（GC/MS）的全-O-三甲基甲硅烷基（TMS）衍生物的单糖甲基糖苷的样品通过酸性甲醇分解。 首先从客户提供的干燥样品中制备甲基糖苷，方法是在80 ℃下在1 M HCl的甲醇溶液中甲醇分解（18-22小时），然后在甲醇中用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（0.5小时）将样品全-O-三甲基甲硅烷基化。[这些程序如先前在Merkle和Poppe（1994）Methods Enzymol. 230：1-15;约克等人（1985）酶学方法（Methods Enzymol. 118：3-40.] TMS甲基糖苷的GC/MS分析在与5975 b MSD连接的HP 6890 GC上进行，使用All Tech EC-1熔融石英毛细管柱（30 m ² 0.25 mm ID）。 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。 最初，将透析后的样品等分试样悬浮在约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20分钟。然后加入碱10分钟，最后加入更多的碘甲烷20分钟。加入更多的碘甲烷和NaOH碱是为了确保聚合物的完全甲基化。样品处理后，通过超氘化物还原全甲基化物质以还原糖醛酸甲酯，然后使用2 M三氟乙酸水解（在密封管中于121 ℃下2 h），用NaBD 4还原，并使用乙酸酐/三氟乙酸乙酰化。 在与5970 MSD（质量选择检测器，电子碰撞电离模式）连接的Hewlett Packard 5890 GC上分析所得PMAA;在30 m Supelco 2330键合相熔融石英毛细管柱上进行分离。 MALDI Ms 将样品溶解在去离子水（1 mg/ml）中，并将1 μ l溶液点在从乙腈/水（1 ∶ 1）中干燥的DHB斑点上，并在Bruker MicroFlex质谱仪上进行MALDI MS，该质谱仪以正离子模式运行。 所有质量均通过在样品前立即运行的麦芽寡糖对照品进行校准。