DETERMINATION OF N-TERMINUS SEQUENCE AND O-LINKED GLYCAN ANALYSIS

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，通过在发酵液中加入氯仿和甲醇，从发酵液中提取脂肪和蛋白质。用丙酮/水溶液进一步洗涤沉淀物四次。用一小份富含蛋白质的粉末(1%)进行SDS-PAGE分析，其余粉末进行b-去除法进行O-糖链分析。B-消除后的溶液通过Dowex树脂脱盐，然后去除硼酸盐和C18净化。这样得到的释放的O-葡聚糖被全甲基化并用质谱仪进行了分析。用于样品分析的详细程序如下所示。 从发酵液中制取富含蛋白粉 根据Aoki.et al(2007)的方法，从发酵液中制备富含蛋白质的粉末。简单地说，通过调整溶剂混合物从发酵液中去除脂肪和沉淀蛋白质，最终得到氯仿/甲醇：水的比例为4：8：3。用离心法收集不溶的蛋白质物质，再提取3次。最后的不溶性蛋白质颗粒用冷丙酮/水(4：1，v/v)进一步洗涤4次，并在氮气气流下干燥。 十二烷基硫酸钠-页面 对一份1%的富蛋白粉末进行了SDS-PAGE分析，以了解富蛋白粉末中的蛋白质含量。样品在4-15%的SDS-PAGE梯度凝胶中分离。电泳后，考马斯亮蓝染色。 O-键合葡聚糖制剂 富含蛋白质的粉末中的O-连接碳水化合物组分通过消除程序被切割。简单地说，向样品中加入1M硼氢化钠(1M)在50 mM氢氧化钠(NaOH)中，并在45oC下孵化过夜。孵化的样品用10%的醋酸中和，通过DowexTM树脂填充柱(50W x 8-100，Sigma Aldrich，St.Louis，MO)脱盐并冷冻干燥。在氮气流动下，用甲醇/冰醋酸(9：1)除去硼酸盐，样品通过C18反相柱。碳水化合物组分(O-连接的多糖)用5%的冰醋酸洗脱。碳水化合物组分通过冷冻干燥，然后根据Anuula和Taylor(Anuula and Taylor，1992)的方法进行全甲基化，并用质谱仪进行分析。 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。通过使用AB SCIEX TOF/TOF“5800系统(应用生物系统)获得光谱。 NSI-MSN分析按照复合碳水化合物研究中心(Aoki et.Al，2007)。在配备纳米级离子源的LTQ Orbitrap XL质谱仪(ThermoFisher)上进行了质量分析。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. Methods: First, lipids were extracted and proteins were precipitated from fermentation solution by adding chloroform and methanol to the solution. The precipitate was further washed with acetone/water solution four times. A small aliquot of protein rich powder (1%) was used for examine the protein powder by SDS-PAGE and the rest of the powder was subjected to b-elimination for O-glycan analysis. The solution after b-elimination was then desalted by passing though Dowex resin, followed by borate removal and C18 clean up. The released O-glycans thus obtained were permethylated and profiled by mass spectrometry. The detailed procedures used for your sample analysis are shown in detail below. Preparation of protein rich powder from fermentation solution Protein rich powder was prepared from fermentation solution according to the method of Aoki.et.al (2007). Briefly, lipids were removed and proteins were precipitated from the fermentation solution by adjusting the solvent mixture to give a final ratio of chloroform/methanol/water equal to 4:8:3. The extract was incubated at room temperature with end-over-end agitation. The insoluble proteinaceous material was collected by centrifugation and re-extracted three times. The final pellet of insoluble protein was further washed with cold-acetone/water (4:1, v/v) four times and dried under a stream of nitrogen. SDS-PAGE An aliquot of protein rich powder (1%) were analyzed by SDS-PAGE to see protein content in protein rich powder. The samples were separated in a 4-15% SDS-PAGE gradient gel. After electrophoresis, the resolved proteins were stained with Coomassie blue. O-linked glycan preparation O-linked carbohydrate fractions were cleaved from protein rich powder by ¿-elimination procedures. Briefly, 1 mL of 1 M sodiumborohydride in 50 mM Sodiumhydroxide (NaOH) were added to the samples and incubated overnight at 45oC. The incubated samples were neutralized with 10%acetic acid and desalted by passing through a packed column of DowexTM resins (50 W x 8--100, Sigma Aldrich, St. Louis,MO) and lyophilized. The borate was removed with methanol/acetic acid (9:1) under a streamof nitrogen gas, and the samples were passed through a C18 reversed phase cartridge. The carbohydrate fractions (O-linked glycans) were eluted with 5% acetic acid. The carbohydrate fractions were 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 AB SCIEX TOF/TOF" 5800 System (Applied Biosystems). NSI-MSn analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki et. al, 2007). Mass analysis was determined 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.