N-LINKED OLIGOSACCHARIDE PROFILING (MASS SPECTROMETRY) OF ONE SAMPLE

一个样品的 N 联寡糖分析（质谱）

• 批准号: 7602825
• 负责人: Parastoo Azadi
• 金额: $ 0.12万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602825
• 关键词: 2-Mercaptoethanol; Acetic Acid; Acetic Acids; Acetylation; Amino Sugars; Blood capillaries; Buffers; Client; Computer Retrieval of Information on Scientific Projects Database; Detection; Digestion; Dimethyl Sulfoxide; Enzymes; Funding; Glycosides; Grant; Heating; Hour; Incubated; Institution; Link; Manufacturer Name; Mass Fragmentography; Mass Spectrum Analysis; Methanol; Methods; Methylene Chloride; Monosaccharides; New England; Nonidet P-40; Oligosaccharides; Peptide N-Glycosidase; Peptide N-glycohydrolase F; Peptides; Procedures; Protocols documentation; Research; Research Personnel; Resources; Sampling; Silicon Dioxide; Sodium Hydroxide; Source; Standards of Weights and Measures; Time; United States National Institutes of Health; Water; Weight; acetic anhydride; capillary; methyl iodide; pyridine; sugar

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. N-linked oligosaccaharides were released from the samples by treatment with peptide Nglycosidase F (PNGase F, N-glycanase) using the enzyme digestion protocol supplied by the manufacturer (New England Biolabs, Beverly,MA). The samples were re-suspended in 200 ml d H2O and 20 ml of 10X denaturing buffer (5% SDS and 10% ¿-mercaptoethanol). The sample and standards were then denatured by heating for 5 minutes at 100¿ C. After cooling, 20 ml of 10X buffer and 40% NP-40 were added. The samples were mixed and 4 ml of enzyme (30 U) were added. Samples were incubated overnight at 37¿C. The digested samples were applied to a C18 classic SEP-PAK. This was eluted with 5 ml of 5% acetic acid to collect the N-linked sugars. The sample fractions were dried and permethylated by the method of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). After permethylation, the samples were extracted three times with methylene chloride/water in order to remove any impurities. The resulting permethylated samples were re-suspended in methanol and analyzed by MALDI-TOF. Glycosyl composition 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 457 mg (client weight) of the dry sample 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 [Merkle, R.K. and Poppe, I. (1994) Methods Enzymol. 230:1-15; York W.S., Darvill, A.G., McNeil, M., Stevenson, T.T., and Albersheim, P. (1985) Methods Enzymol. 118:3-40]. GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm ID).

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。 使用制造商（New England Biolabs，Beverly，MA）提供的酶消化方案，通过肽 N 糖苷酶 F（PNGase F，N-聚糖酶）处理，从样品中释放 N-连接寡糖。 200 ml d H2O 和 20 ml 10X 变性缓冲液（5% SDS 和 10% ¿-巯基乙醇）。然后将样品和标准品在 100°C 下加热 5 分钟使其变性。 C. 冷却后，加入 20 ml 10X 缓冲液和 40% NP-40，将样品混合，并加入 4 ml 酶（30 U），将样品在 37℃下孵育过夜。 C. 将消化的样品应用于 C18 经典 SEP-PAK，用 5 ml 5% 乙酸洗脱以收集 N-连接糖。 将样品级分按照Ciukanu 和Kerek (1984) CarboHydr. 131:209-217 的方法进行干燥和全甲基化（用干燥DMSO 中的氢氧化钠和碘甲烷处理），然后用亚甲基将样品萃取3 次。将所得全甲基化样品重新悬浮在甲醇中并通过 MALDI-TOF 进行分析。 糖基组成 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。 首先通过在 1 M HCl 的甲醇溶液中在 80° 下进行甲醇分解，从 457 mg（客户重量）的干燥样品中制备甲基糖苷。 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）。这些程序如先前所述进行[Merkle, R.K. 和 Poppe, I. (1994)Methods Enzymol. 118:3-40] 使用 Supelco 在连接至 5970 MSD 的 HP 5890 GC 上进行 TMS 甲基糖苷的 GC/MS 分析。 DB-1 熔融石英毛细管柱（30m x 0.25 mm ID）。