O-LINKED OLIGOSACCHARIDE PROFILING OF 3 SAMPLES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 ？-除盐、除盐、除硼 O-连接的碳水化合物部分通过消除程序从糖蛋白中分离出来。简而言之，250岁的L 将MM NaOH加入到每个样品中，然后检查pH值。在确定pH值为碱性时， 另将含有19毫克硼氢化钠的50 mM氢氧化钠加入到样品中，旋转，然后 在450摄氏度下孵化过夜。然后用10%的冰醋酸中和孵化的样品，并用 通过DOWEXTM树脂填充柱(50W×8100，Sigma Aldrich)，然后进行冷冻干燥。干的 样品在全甲基化前用甲醇：冰醋酸(9：1)在氮气中清除。 全O-甲基化碳水化合物的制备及C18的净化 碳水化合物部分溶解在二甲基亚砜中，然后用氢氧化钠和碘甲烷甲基化 (Ciucanu和Kerek，1984)。反应通过加水来熄灭，全氧甲基化的碳水化合物 用二氯甲烷提取。进一步清除了Per-O-甲基化多糖中的污染物。简而言之，这些多糖 装入C18Sep Pak色谱柱，然后用纳米纯水和15%乙腈洗涤。多聚糖 然后用85%乙腈洗脱。纯化的多聚糖在氮气气流下干燥并溶解 在用质谱仪进行分析之前用甲醇。 基质辅助激光解吸飞行时间质谱仪(MALDI-TOF) MALDI/TOF-MS在反射镜正离子模式下进行，使用2-二羟基苯甲酸(DHBA，20 mg/mL溶液 在50%甲醇：水中)作为基质。使用4700蛋白质组学分析仪(已应用)获得所有光谱 生物系统)。 电喷雾电离线性离子陷阱质谱仪(ESI-LCQ/MSN) LC-MS(Thermo Finnigan)的质量分析是通过直接输注溶解在1，2，3，6，6，6，6，6，6，6，6，6，6，6，6，6，6-三甲基的过甲基化多聚糖进行的。 用50%甲醇中的氢氧化钠(5pmol/L)通过注射器以恒定流速1？L/分钟进入LC-Q仪器 Pump(哈佛仪器)。毛细管温度设置为200oC，在阳离子中进行MS分析 模式。在MS/MS中，碰撞能被设置为35%的碎裂。

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. ¿- elimination, Desalting, Borate removal O-linked carbohydrate fractions were cleaved from the glycoprotein by ¿-elimination procedures. Briefly, 250 ¿L of 50 mM NaOH were added to each of the samples and then checked for pH. Upon determination that the pH was basic, another 250 ¿L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas before permethylation. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and were dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF) MALDI/TOF-MS was performed in the reflector positive ion mode using ¿-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol:water) as a matrix. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). ElectroSpray Ionization  Linear Ion Trap mass Spectrometry (ESI-LCQ/MSn) Mass analysis by LCQ-MS (Thermo Finnigan) was performed by direct infusion of permethylated glycans dissolved in 1 mM NaOH in 50% methanol ( 5 pmol/¿L) into the LCQ instrument at a constant flow rate of 1 ¿L/min via a syringe pump (Harvard Apparatus). The capillary temperature was set to 200oC and MS analysis was performed in positive ion mode. The collision energy was set at 35% for fragmentation in MS/MS.