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： 每个样品(100？g)的等量用于N-连锁图谱分析。样品首先变性，用胰酶消化，然后用C18Sep-pak富集糖多肽。然后用PNGase F从糖肽中酶解释放N-糖链，用C18-Sep-pak从残留物中分离释放的N-糖链，并在进行质谱分析之前进行全甲基化。 用于样品分析的详细程序如下所示。 N-连接葡聚糖制剂 每个样品干燥一等分(100？g)，用0.1M Tris-HCl缓冲液重配制，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小柱，释放的N-糖链用5%的冰醋酸洗脱，冷冻干燥，然后根据Anumula和Taylor(Anuula and Taylor，1992)的方法进行全甲基化，并进行质谱分析。 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: Aliquot of each sample (100¿g) were used for N-linked profiling. Samples were denatured first and digested with trypsin, followed by glycopeptides enrichment by C18 sep-pak. N-glycans were then released from the glycopeptides enzymatically by PNGase F. Released N-glycans were separated from residual contents by C18 sep-pak and permethylated prior to mass spec analysis. Detailed procedures used for your sample analysis are shown in detail below. N-linked glycan preparation An aliquot (100¿g) of each sample was dried and reconstituted with 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 released N-glycans was eluted with 5% acetic acid and dried by lyophilization, and then permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) 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).