N-LINKED OLIGOSACCHARIDE PROFILING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 首先，对样品进行变性，然后用胰酶消化，然后进行糖肽浓缩。然后用PNGase F酶促释放N-连接的多聚糖，释放的多聚糖用C18Sep-pak分离和全甲基化，然后进行质谱分析。用于样品分析的详细程序如下所示。 N-连接葡聚糖的释放 样品溶于0.1M Tris-HCl缓冲液中，pH 8.2，含0.01M CaCl2。然后将样品在100℃加热5分钟变性，冷却后，用胰酶(37℃，过夜)消化样品。然后将样品在100℃加热5分钟以使胰酶失活。然后将样品通过C18 Sep-pak色谱柱，并用5%的冰醋酸洗涤以去除污染物(盐、游离糖等)。多肽和糖肽用20%异丙醇5%冰醋酸、40%异丙醇5%冰醋酸和100%异丙醇串联洗脱，在高速真空浓缩器中干燥。将干燥的样品与PNGase F在37℃下孵育一夜，释放N-糖链。消化后，样品通过C18Sep-pak色谱柱，碳水化合物部分用5%的冰醋酸洗脱并冷冻干燥。 全O-甲基化碳水化合物的制备 将碳水化合物部分溶解在二甲基亚砜中，然后根据Anuula和Taylor的方法进行全甲基化(Anuula和Taylor，1992)。加水使反应熄灭，用二氯甲烷提取全氧甲基化碳水化合物。进一步清除了Per-O-甲基化多糖中的污染物。简而言之，将多聚糖装入C18Sep pak色谱盒，然后用纳米纯水和15%乙腈洗涤。然后用85%的乙腈洗脱多糖。纯化的多聚糖在氮气气流下干燥，用甲醇溶解，并用质谱仪分析。 质谱学 MALDI/TOF-MS色谱柱为反射镜正离子模式，以二羟基苯甲酸(DHBA，20 mg/mL甲醇：水)为基质。光谱是用Microflex LRF(Bruker)获得的。

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: First of all, the sample was denatured and then digested with Trypsin followed by glycopeptides enrichment. N-linked glycans were then released enzymatically by PNGase F. Released glycans were separated by C18 sep-pak and permethylated prior to mass spec analysis. Detailed procedures used for your sample analysis are shown in detail below. Release of N-linked glycans The sample was dissolved in 0.1 M Tris-HCl buffer, pH 8.2 containing 0.01 M CaCl2. The sample then was denatured by heating for 5 minutes at 100¿C. After cooling, the sample was digested with the trypsin (37oC, overnight). The sample was then heated at 100¿ C for 5 min to inactivate trypsin. The sample was then passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, free sugar, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and incubated with PNGase F at 37¿ C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Preparation of the per-O-methylated carbohydrates 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 and were dissolved with methanol and profiled by mass spectrometry. Mass spectrometry 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. The spectrum was obtained by using a Microflex LRF (Bruker).