LINKAGE ANALYSIS BY GC-MS

通过 GC-MS 进行连锁分析

基本信息

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
项目类别：
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

项目摘要

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.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供该子项目的主要支持。并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源的子项目可能列出的总成本。代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。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 截止膜；G BioSciences）中，在 4℃ 下对 4 L 纳米纯水进行透析约 24 小时，以去除盐和其他污染物。整个透析期间更换纳米纯水四次。透析后，将样品冻干并最终合并在一根微量离心管中用于酶。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 摄氏度加热块中 5 分钟，使蛋白质变性。冷却至室温后，用 6 ¿ 处理样品。 g 胰蛋白酶和 6 ¿ g 胰凝乳蛋白酶并在 37℃ 下孵育 18 小时，然后将管置于 100 摄氏度加热块中 5 分钟，使胰蛋白酶和胰凝乳蛋白酶失活。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。一旦装入柱，用 5% 乙酸清洗吸附的样品，并用 20% 异丙醇连续洗脱糖肽/肽。 5% 乙酸、40% 异丙醇、5% 乙酸和 100%洗脱液首先在氮气流下干燥以蒸发异丙醇并最终冻干。将干燥的酶消化物用 50 mM NaPO4 缓冲液（pH~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，样品通过。通过 C18 sep pak 柱，N-连接聚糖级分用 5% 乙酸洗脱并冻干。 N-连接聚糖的全-O-甲基化按照Anumula和Taylor (1992)的方法，将PNGase-F从样品中释放的N-连接聚糖进行全甲基化。简言之，将干燥的洗脱液用二甲亚砜溶解，然后用NaOH和碘甲烷进行甲基化。用二氯甲烷提取水和全O-甲基化聚糖，完成全甲基化对于连锁分析至关重要，因此需要一小部分。通过 MALDI-TOF-TOF MS (Applied Biosystems 4700) 分析全甲基化聚糖以评估概况。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 连接聚糖中制备 PMAA。简言之，用 HCl:水:乙酸（0.5:1.5:8，体积比）在 80℃ 下水解 18 小时，然后水解。水解和还原步骤后，部分甲基化的糖醇的游离羟基被乙酰化。乙酸酐:吡啶 (1:1, v/v) 在沸水中加热 15 分钟以产生 PMAA。气相色谱-质谱联用仪 (GC-MS) PMAA 在与 5970 MSD 连接的 Hewlett Packard 5890 GC 上进行分析。分离在 30 m EC 1 键合相熔融石英毛细管柱 (Altech) 上进行。在以下条件下获得：烤箱温度，140° C (2.0°C/分钟) ¿ 220° C (20°C/分钟) ¿ 300€ C（20°C/分钟）；检测器温度，280°入口温度，250°C； C.