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。 &lt；凝胶内消化&gt； 考马斯蓝染色的凝胶切片切成小块(~1mm3)，用40 mM碳酸氢铵(AmBic)和100%乙腈交替脱色，直到颜色变得透明。去掉的凝胶在10 mM DTT中55℃下于40 mM Ambic中重新溶胀1小时。DTT溶液与55 mM碘乙酰胺(IAM)交换，在黑暗中孵育45min。孵育后用40 mM AmBic和100%乙腈交替洗涤2次。首先用胰酶溶液(40 mM Ambic中的胰酶)在冰上复温45min，然后在37℃下进行蛋白质消化过夜。将上清液转移到另一试管中。用20%乙腈-5%甲酸、50%乙腈-5%甲酸、80%乙腈-5%甲酸串联提取多肽和糖肽。将样品溶液干燥并合并到一个试管中。 &lt；葡聚糖制剂&gt； 提取的胰酶消化液通过C18Sep-pak小柱，并用5%的冰醋酸洗涤以去除污染物(盐、十二烷基硫酸钠等)。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。将干燥的样品合并，然后用50 mM的磷酸二氢钠缓冲液(pH 7.5)重置，在100℃加热5min以灭活胰酶。胰酶消化液与PNGase F在37℃下孵育一夜，以释放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。根据Anuula和Taylor(Anuula and Taylor，1992)的方法对释放的N-连接低聚糖进行全甲基化，并用质谱仪进行分析。 &lt；质谱学&gt； 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。所有光谱均使用Microflex LRF(Bruker)获得。 在装有纳米螺旋离子源的LTQ Orbitrap XL质谱仪(ThermoFisher)上进行了NSI-MSN分析。将全甲基化糖溶解于1 mM氢氧化钠中50%的甲醇中，以0.5？L/分钟的恒定流速直接进入仪器。在30 000分辨率下收集了完整的FTMS光谱。毛细管温度210oC，正离子模式进行MS分析。对于全离子图谱(自动MS/MS分析)，在与前一个窗口重叠2个质量单位的连续2.8个质量单位窗口中，用ITMS模式扫描m/z范围800至2000。

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. <In-gel digestion> Coomassie blue-stained gel slices were cut into smaller pieces (~1 mm3) and destained alternately with 40mM Ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. Destained gel was reswelled in 10 mM DTT in 40mM Ambic at 55¿ C for 1 hr. The DTT solution was exchanged with 55mM Iodoacetamide (IAM) and incubated in the dark for 45 min. Incubation was followed by washing alternately with 40mM AmBic and 100% acetonitrile twice. Dehydrated gel was reswelled with trypsin solution (trypsin in 40 mM Ambic) on ice for 45 min initially, and protein digestion was carried out at 37¿ C overnight. The supernatant was transferred into another tube. Peptides and the glycopeptides were extracted from the gel in series with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The sample solutions were dried and combined into one tube. <Glycan preparation> Extracted tryptic digest was passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, SDS, 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 then reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and heated at 100¿ C for 5 min to inactivate trypsin. The tryptic digest was 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. Released N-linked oligosaccharides were 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. All spectra were obtained by using a Microflex LRF (Bruker). NSI(nanospray ionization)-MSn analysis was performed by using on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. Permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument at a constant flow rate of 0.5 ¿L/ min. A full FTMS spectrum was collected at 30 000 resolution. The capillary temperature was set at 210oC and MS analysis was performed in the positive ion mode. For total ion mapping (automated MS/MS analysis), m/z range, 800 to 2000 was scanned with ITMS mode in successive 2.8 mass unit windows that overlapped the preceding window by 2 mass units.