O-LINKED GLYCOSYLATION SITE MAPPING

O-连接糖基化位点图谱

• 批准号: 8361830
• 负责人: Parastoo Azadi
• 金额: $ 0.18万
• 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: 2011-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361830
• 关键词: Acetonitriles; Algorithms; Blood capillaries; Cysteine; Data; Formic Acids; Funding; Grant; Incubated; Iodoacetamide; Ions; Label; Link; Maps; Methionine; Methods; Modification; National Center for Research Resources; Nitrogen; Peptide Hydrolases; Peptides; Phase; Plant Resins; Principal Investigator; Proteome; Reaction; Recombinants; Research; Research Infrastructure; Resources; Sampling; Serine; Site; Source; Speed; Trifluoroacetic Acid; Trypsin; United States National Institutes of Health; Water; ammonium bicarbonate; capillary; chymotrypsin; cost; glycosylation; ion source; mass spectrometer; oxidation; pressure; triethylamine

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: O-Glycosylation Site Mapping With B-elimination followed by Michael addition (BEMAD) and LC-MS/MS One hundred micrograms of NG or SA was reduced with 5 mM DTT for 1 h at 55 ¿C and carboxyamidomethylated with 15 mM iodoacetamide in the dark for 45 min. The dried dialyzed sample was resuspended in 50 mM ammonium bicarbonate (NH4HCO3) and digested with 5 ¿g of trypsin or chymotrypsin at 37 ¿C for 20 h. Following deactivation of protease at 100 ¿C for 5 min, the sample was dried in a Speed Vac. Dried peptides were then B-eliminated and subjected to Michael addition with DTT via resuspension in 1% triethylamine, 0.1% NaOH, and 10 mM DTT. The reaction was incubated at 42 ¿C for 3 h, and the reaction was quenched with 1% trifluoroacetic acid. The labeled peptides were clean up by reverse phase C18 columns, eluted in 0.1 % formic acid, 80 % acetonitrile, and dried in a Speed Vac. LC-MS/MS analysis was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. The labeled peptides were resuspended with 200 ¿L of mobile phase A (0.1% formic acid in water). The sample was then loaded onto a nanospray tapered capillary column/emitter (360x75x15 ¿m, PicoFrit, New Objective, Woburn, MA) self-packed with C18 reverse-phase resin (10.5 cm, Waters, Milford, MA) in a Nitrogen pressure bomb for 5 min at 1,000 psi (~5 uL load) and then separated via a 160 min linear gradient of increasing mobile phase B at a flow rate of~500 nL/min directly into the mass spectrometer. The resulting data were searched against the recombinant NG or SA sequence using the TurboSequest algorithm (Proteome Discoverer 1.1, Thermo Scientific). The SEQUEST parameters were set to allow 2 Da of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Digested peptides were allowed with up to two missed internal cleavage sites, and the differential modifications of 57.02146 Da, 15.9949 Da and 136.002 Da were allowed for alkylated cysteine, oxidation of methionines and DTT-labeled serine or theonine, respectively.

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： O-糖基化位点定位，先进行 B 消除，然后进行 Michael 加成 (BEMAD) 和 LC-MS/MS 使用 5 mM DTT 在 55 ¿ 1 小时内减少 100 微克 NG 或 SA C 并在黑暗中用 15 mM 碘乙酰胺进行羧酰胺甲基化 45 分钟 将干燥的透析样品重悬于 50 mM 碳酸氢铵 (NH4HCO3) 中并用 5 ¿ g 胰蛋白酶或胰凝乳蛋白酶，37 ¿ C 20 小时后，在 100 ¿ C 5 分钟，将样品在 Speed Vac 中干燥，然后将干燥的肽进行 B-消除，并通过在 1% 三乙胺、0.1% NaOH 和 10 mM DTT 中重悬进行 Michael 加成反应。 42° C反应3小时，用1%三氟乙酸淬灭反应。标记的肽通过反相C18柱净化，用0.1%甲酸、80%乙腈洗脱，并在Speed Vac中干燥。 LC-MS/MS 分析在配备纳喷雾离子源的 LTQ Orbitrap Discoverer 质谱仪 (Thermo Scientific) 上进行，标记的肽用 200 ¿然后将 L 流动相 A（0.1% 甲酸水溶液）上样至使用 C18 反相树脂自装填的纳喷雾锥形毛细管柱/发射器（360x75x15 µm，PicoFrit，New Objective，Woburn，MA）。 （10.5 cm，Waters，Milford，MA）在氮气压力弹中以 1,000 psi (~5 uL负载），然后通过 160 分钟线性梯度增加流动相 B，以约 500 nL/min 的流速直接进入质谱仪。使用 TurboSequest 算法（Proteome）针对重组 NG 或 SA 序列搜索所得数据。 Discoverer 1.1，Thermo Scientific)。 SEQUEST 参数设置为允许 2 Da 的前体离子质量容差和 0.8 Da 的单同位素质量碎片离子容差。消化的肽最多允许有两个缺失的内部切割位点，并且分别允许烷基化半胱氨酸、甲硫氨酸氧化和 DTT 标记的丝氨酸或茶氨酸进行 57.02146 Da、15.9949 Da 和 136.002 Da 的差异修饰。