O-LINKED OLIGOSACCHARIDE PROFILING

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： 还原消除法制备O-聚糖 将500 μ g来自C33 A和RP 215的PNGaseF消化的糖肽在含有1.0 M硼氢化钠的100 mM NaOH中于45 ℃下进行碱性还原消除18 h。将反应混合物用10%乙酸中和，并在DOWEXTM树脂柱（50 W × 8）上脱盐100，Sigma Aldrich）。将用5%乙酸洗脱的物质冻干，用甲醇蒸发除去硼酸。通过Sep-Pak C18柱纯化释放的O-聚糖。 全-O-甲基化聚糖的制备 将聚糖组分溶于二甲亚砜中，然后根据Anumula和Taylor的方法（Anumula和Taylor，1992）进行全甲基化。通过加入水淬灭反应，用二氯甲烷萃取全-O-甲基化的碳水化合物。在氮气流下干燥全-0-甲基化聚糖。通过Sep-Pak C18柱纯化全甲基化O-聚糖。 纳米喷雾电离-线性离子阱质谱 在配备有纳米喷雾离子源的LTQ Orbitrap Discoverer质谱仪（Thermo Scientific）上通过NSI-MS进行质量分析。简而言之，将全甲基化聚糖溶于1 mM NaOH的50%甲醇溶液中，并使用纳米喷雾源以0.5 L/min的注射器流速直接注入线性离子阱质谱仪中。毛细管温度设定为210 C，并在正离子模式下进行MS分析。在MS/MS模式下通过CID进行裂解，应用25%碰撞能量。使用Domon和Costello的命名法（Domon和Costello，1988）指导MS/MS谱图中碎片的描述。

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: Preparation of O-glycans by Reductive ¿- Elimination Five hundred microgram of PNGaseF digested glycopeptides from C33A and RP215 were subjected to alkaline reductive elimination in 100 mM NaOH containing 1.0 M sodium borohydride at 45 ¿C for 18 h. The reaction mixture was neutralized with 10% acetic acid and desalted on a column of DOWEXTM resins (50W x 8100, Sigma Aldrich). The material eluting with 5 % acetic acid was lyophilized and boric acid was removed by evaporation with methanol. Released O-glycans were purified by Sep-Pak C18 cartridge column. Preparation of the per-O-methylated glycans The glycan fraction was dissolved in dimethylsulfoxide and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were dried under a stream of nitrogen. Permethylated O-glycans were purified by Sep-Pak C18 cartridge column. Nanospray Ionization-Linear Ion Trap Mass Spectrometry Mass analysis by NSI-MS was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer using a nanospray source at a syringe flow rate of 0.5 ¿l/min. The capillary temperature was set to 210 ¿C, and MS analysis was performed in positive ion mode. Fragmentation by CID in MS/MS modes 25% collision energy was applied. The nomenclature of Domon and Costello (Domon and Costello, 1988) was used to guide the depiction of fragmentation derived from MS/MS spectra.