O-LINKED OLIGOSACCHARIDE PROFILING AND LINKAGE ANALYSIS OF ONE SAMPLE

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。 通过C18 sep-pak滤芯进行除盐、脱盐、去除硼酸盐、清洁 O-连接的碳水化合物部分通过消除程序从糖肽中裂解。 简言之，向样品中加入溶于50 mM氢氧化钠（NaOH）的1 M硼氢化钠（NaBH 4），并在45 ℃下孵育17 h。 然后将温育的样品用10%乙酸中和，并通过DOWEXTM树脂的填充柱（50 W × 8）脱盐  100，Sigma Aldrich），然后冻干。 在氮气流下用甲醇：乙酸（9：1，v/v）清洗干燥样品的硼酸盐。 然后使样品通过C18反相柱以纯化O-聚糖。 首先用5%乙酸洗脱碳水化合物级分（O-连接的聚糖），然后用20%异丙醇的5%乙酸溶液、40%异丙醇的5%乙酸溶液和100%异丙醇将肽依次洗脱至微量离心管中。 通过冻干干燥碳水化合物级分，而在高速真空浓缩器中干燥肽级分，然后在一个管中合并。 全-O-甲基化碳水化合物的制备，通过C18 sep-pak柱净化 将碳水化合物部分溶于二甲亚砜中，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。 进一步清除全-0-甲基化聚糖的污染物。 简言之，将聚糖上样至C18 sep-pak柱中，然后用纳米纯水和15%乙腈洗涤。 然后用85%乙腈洗脱聚糖。 在氮气流下干燥纯化的聚糖。 糖基连接分析 为了测定糖键，从全甲基化的0-聚糖制备部分甲基化的糖醇乙酸酯。 简言之，在100 ℃下用2 M TFA水解全甲基化聚糖4 h，然后用30 mM NaOH中的1% NaBH 4还原，并在100 ℃下用乙酸酐/吡啶（1：1，v/v）乙酰化15 min。通过GC-MS分析由此获得的部分甲基化糖醇乙酸酯。 基质辅助激光解吸飞行时间质谱 MALDI/TOF-MS以反射器正离子模式进行，使用？-二羟基苯甲酸（DHBA，20 mg/mL在50%甲醇：水中的溶液）作为基质。 通过使用4700蛋白质组学分析仪（Applied Biosystems）获得所有光谱。 气相色谱-质谱（GC-MS） 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上分析部分甲基化的糖醇乙酸酯。 分离在两个不同的柱上以不同的温度进行，如下所示：

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, Cleaning by C18 sep-pak cartridge O-linked carbohydrate fractions were cleaved from the glycopeptides by ¿-elimination procedures. Briefly, 1M sodium borohydride (NaBH4) in 50 mM Sodiumhydroxide (NaOH) was added to the sample and incubated 17 h at 45oC. The incubated sample then was neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8  100, Sigma Aldrich) and then was lyophilized. Dried sample was cleaned of borate with methanol:acetic acid (9:1, v/v) under a stream of nitrogen gas. Then the sample was passed through a C18 reversed phase cartridge to purify the O-glycans. The carbohydrate fractions (O-linked glycans) were first eluted with 5% acetic acid and then the peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The carbohydrate fraction was dried by lyophilization, whereas the peptide fractions were dried in the speed vacuum concentrator and then combined in one tube. Preparation of the per-O-methylated carbohydrates, Cleaning up by C18 sep-pak cartridge The carbohydrate 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 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. Glycosyl linkage analysis For determination of sugar linkages, partially methylated alditol acectates were prepared from permethylated O-glycans. Briefly, permethylated glycans were hydrolysed with 2M TFA at 100oC for 4 h, followed by reduction with 1% NaBH4 in 30mM NaOH and acetylation with acetic anhydride/pyridine (1:1, v/v) at 100 ¿C for 15 min. The partially methylated alditol acetates thus obtained were analyzed by GC-MS. 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). Gas Chromatograph-Mass Spectrometry (GC-MS) The partially methylated alditol acetates were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separations were performed on two different columns with different temperature as follows;