LINKAGE ANALYSIS BY GC-MS

通过 GC-MS 进行连锁分析

• 批准号: 8363052
• 负责人: Parastoo Azadi
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363052
• 关键词: 1-Propanol; Acetates; Acetic Acids; Aliquot; Blood capillaries; Buffers; Dialysis procedure; Digestion; Dimethyl Sulfoxide; Electrons; Enzymes; Funding; Furuncles; Gases; Glycopeptides; Grant; Heating; Hour; Hydrolysis; Hydroxyl Radical; Incubated; Link; Mass Spectrum Analysis; Membrane; Methods; Methylation; Methylene Chloride; National Center for Research Resources; Nitrogen; Peptide N-Glycosidase; Peptide N-glycohydrolase F; Peptides; Phase; Polysaccharides; Preparation; Principal Investigator; Propanols; Proteins; Reaction; Research; Research Infrastructure; Resources; Salts; Sampling; Sep-Pak C18; Series; Silicon Dioxide; Solutions; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stream; Sugar Alcohols; Technology; Temperature; Time; Trypsin; Tube; United States National Institutes of Health; Water; acetic anhydride; ammonium bicarbonate; capillary; chymotrypsin; cost; detector; genetic linkage analysis; ionization; methyl iodide; pyridine

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: Dialysis The sample solution (~6 mL) was transferred into 3 Tube-O-Dialyzers (4.0 kDa cut-off membrane; G BioSciences). Dialysis was performed against 4 L of nanopure water at 4oC for about 24 hours to remove salts and other contaminants. Nanopure water was replaced four times during the entire dialysis period. After dialysis, the sample was lyophilized and eventually combined in one microcentrifuge tube for enzyme digestion. Release of N-linked glycans The dried sample was dissolved with 50 mM ammonium bicarbonate and the tube was placed in a 100-degree Celsius heating block for 5 min to denature the protein. After cooling to room temperature, the sample was treated with 6 ¿g of trypsin and 6 ¿g of chymotrypsin and incubated at 37oC for 18 hr. Trypsin and chymotrypsin were deactivated thereafter by placing the tube in a 100-degree Celsius heating block for 5 min. The tryptic/chymotryptic digest was cleaned of any contaminants by passing it through a C18 sep pak cartridge. Once loaded in the cartridge, adsorbed sample was cleaned with 5% acetic acid and the glycopeptides/peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid, and 100% iso-propanol. The eluate was dried initially under a stream of nitrogen to evaporate the iso-propanol and lyophilized eventually. The dried enzyme digest was dissolved with 50 mM NaPO4 buffer (pH~7.5), treated with PNGase F and incubated at 37oC for 18 hr to release the N-linked glycans. At the end of the N-glycanase digestion, the sample was passed through a C18 sep pak cartridge and N-linked glycans fraction was eluted with 5% acetic acid and lyophilized. Per-O-methylation of N-linked Glycans The PNGase-F released N-linked glycans from the sample was permethylated following the methods of Anumula and Taylor (1992). Briefly, the dried eluate was dissolved with dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water and per-O-methylated glycans were extracted with methylene chloride. Completion of permethylation is critical for linkage analysis, hence, a small aliquot of the permethylated glycans was analyzed by MALDI-TOF-TOF MS (Applied Biosystems 4700) to evaluate the profile. Preparation of Partially Methylated Alditol Acetates (PMAAs) For the determination of glycosyl linkages, PMAAs were prepared from the released permethylated N-linked glycans. Briefly, permethylated glycans were hydrolyzed with HCl:Water:Acetic acid (0.5:1.5:8, by vol.) at 80oC for 18h, followed by reduction with NaBD4. After hydrolysis and reduction steps, the free hydroxyls of the partially methylated alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) in boiling water for 15 min to produce PMAAs. Gas Chromatograph-Mass Spectrometry (GC-MS) The PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode). The separation was performed on a 30 m EC 1 bonded phase fused silica capillary column (Altech). Electron impact mass spectra were obtained under the following conditions: oven temperature, 140¿C (2.0¿C/min) ¿ 220¿C (20¿C/min) ¿ 300¿C (20¿C/min); detector temperature, 280 ¿C; inlet temperature, 250 ¿C.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 研究方法： 透析 将样品溶液（约6 mL）转移至3个Tube-O-透析器（4.0 kDa截留膜; G BioSciences）中。 在4 ° C下对4L纳米纯水进行透析约24小时以除去盐和其他污染物。 在整个透析期间，纳米纯水更换了四次。 透析后，将样品冻干，并最终在一个微量离心管中合并用于酶消化。 N-连接聚糖的释放 将干燥的样品用50 mM碳酸氢铵溶解，并将管置于100摄氏度的加热块中5分钟以使蛋白质变性。 冷却至室温后，用6 μ g胰蛋白酶和6 μ g胰凝乳蛋白酶处理样品，并在37 ℃下孵育18小时。然后将试管置于100 ℃加热块中5分钟，使胰蛋白酶和胰凝乳蛋白酶失活。 通过使胰蛋白酶/胰凝乳蛋白酶消化物通过C18 sep pak柱清除任何污染物。 一旦加载到柱中，用5%乙酸清洁吸附的样品，并用5%乙酸中的20%异丙醇、5%乙酸中的40%异丙醇和100%异丙醇连续洗脱糖肽/肽。 首先在氮气流下干燥物以蒸发异丙醇并最终冻干。 将干燥的酶消化物用50 mM NaPO 4缓冲液（pH约7.5）溶解，用PNGase F处理，并在37 ℃下孵育18小时以释放N-连接聚糖。 在N-聚糖酶消化结束时，使样品通过C18 sep pak柱，用5%乙酸洗脱N-连接聚糖级分并冻干。 N-连接聚糖的全-O-甲基化 按照Anumula和Taylor（1992）的方法，对从样品中释放的PNGase-F N-连接聚糖进行全甲基化。 简言之，将干燥的水用二甲基亚砜溶解，然后用NaOH和碘甲烷甲基化。 通过加入水淬灭反应，并用二氯甲烷萃取全-O-甲基化聚糖。 全甲基化的完成对于连锁分析至关重要，因此，通过MALDI-TOF-TOF MS（Applied Biosystems 4700）分析小等份的全甲基化聚糖，以评价图谱。 部分甲基化糖醇乙酸酯（PMAA）的制备 为了测定糖基键，从释放的全甲基化N-连接聚糖制备PMAA。 简言之，用HCl：水：乙酸（0.5：1.5：8，体积比）水解全甲基化聚糖。在80 ° C下反应18小时，然后用NaBD 4还原。 在水解和还原步骤之后，将部分甲基化的糖醇的游离羟基用乙酸酐：吡啶（1：1，v/v）在沸水中乙酰化15分钟以产生PMAA。 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PMAA。 在30 μ m EC 1键合相熔融石英毛细管柱（Altech）上进行分离。 在以下条件下获得电子轰击质谱：烘箱温度，140 ℃（2.0 ℃/min）<$220 ℃（20 ℃/min）<$300 ℃（20 ℃/min）;检测器温度，280 ℃;入口温度，250 ℃。