GLYCOSYL COMPOSTION ANALYSIS AND N-LINKED PROFILING OF ONE SAMPLE

一个样品的糖基组成分析和 N 连锁分析

• 批准号: 7602829
• 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/7602829
• 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; Endoglycosidase F; Enzymes; Funding; Glycosides; Grant; Heating; Hour; Incubated; Institution; Link; Manufacturer Name; Mass Fragmentography; Methanol; Methods; Methylene Chloride; Monosaccharides; New England; Nonidet P-40; Peptide N-Glycosidase; Peptide N-glycohydrolase F; 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 N glycosidase F (PNGase F, N-glycanase) using the enzyme digestion protocol supplier by the manufacturer (New England Biolabs, Beverly,MA). The samples were resuspended in 200¿l d H2O and 20¿l 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¿l of 10X buffer and 40% NP-40 were added. The samples were mixed, and 4¿l of enzyme (30U) 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 resuspended 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 ¿g (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（新英格兰生物实验室，贝弗利，马萨诸塞州）提供的酶消化方案，用肽 N 糖苷酶 F（PNGase F，N-聚糖酶）处理，从样品中释放 N-连接寡糖。样品在 200°C 中重悬。 l d H2O 和 20¿ 1 升 10X 变性缓冲液（5% SDS 和 10% 巯基乙醇）然后在 100° 下加热 5 分钟使样品和标准品变性。 C.冷却后，20¿添加l 10X缓冲液和40% NP-40，将样品混合，并加入4¿添加l酶(30U)，将样品在37°温育过夜。 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。 甲基糖苷首先由 457 ¿ g（客户重量）干样品，在 80° 的 1 M HCl 甲醇溶液中进行甲醇分解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。 ¿这些程序如前所述进行（Merkle，R.K.和 Poppe，I.（1994）Methods Enzymol.230：1-15；York W.S.，Darvill，A.G.，McNeil，M.，Stevenson，T.T. ，和Albersheim，P.（1985）酶方法118：3-40）。使用 Supelco DB-1 熔融石英毛细管柱（30m x 0.25 mm ID）在连接至 5970 MSD 的 HP 5890 GC 上对 TMS 甲基糖苷进行 GC/MS 分析。