PROTEIN IDENTIFICATION AND O-GLYCOSYLATION SITE MAPPING

蛋白质鉴定和 O-糖基化位点定位

• 批准号: 8363081
• 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/8363081
• 关键词: Acetonitriles; Algorithms; Blood capillaries; Complementary DNA; Cysteine; Data; Formic Acids; Funding; Grant; Incubated; Iodoacetamide; Ions; Label; Maps; Methionine; Methods; Modification; National Center for Research Resources; Nitrogen; Peptide Hydrolases; Peptides; Phase; Plant Resins; Principal Investigator; Proteins; Proteome; Reaction; Recombinants; Research; Research Infrastructure; Resources; Sampling; Serine; Site; Source; Speed; Technology; Trifluoroacetic Acid; Trypsin; United States National Institutes of Health; Water; ammonium bicarbonate; capillary; 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: B-elimination followed by Michael addition (BEMAD) for O-Glycosylation site mapping One hundred micrograms of CBH2b-cDNA and CBH2b-YO were 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 samples were resuspended in 50 mM ammonium bicarbonate (NH4HCO3) and digested with 5 ¿g of trypsin at 37 ¿C for 20 h. Following deactivation of protease at 100 ¿C for 5 min, the samples were digested with 5 ¿g of Glu-C at 25 ¿C for 20 h and then dried down 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. Protein identification by LC-MS/MS 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 CBH2b-cDNA or CBH2b-YO 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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 方法： b-灭绝，然后是迈克尔（Bemad）（bemad）进行O-糖基化位点映射 在55 c下用5 mM DTT用5 mM DTT降低100微克的CBH2B-CDNA和CBH2B-YO，并在黑暗中用15 mM iodoacetamide在黑暗中用15 mM的羧甲基化45分钟。将干燥的透析样品重悬于50毫米碳酸氢铵（NH4HCO3）中，并在37 c时用5？g的胰蛋白酶消化20小时。在100 c下蛋白酶停用5分钟后，将样品用5 g glu-c在25°C下消化20小时，然后在速度VAC中干燥。然后对干胡椒粉进行B-释放，并通过在1％三乙胺，0.1％NaOH和10 mM DTT中重悬于DTT中加入Michael。将反应在42°C下孵育3小时，并用1％三氟乙酸淬灭反应。标记的肽通过反向相C18柱进行清理，在0.1％甲酸，80％乙腈中洗脱，并在速度VAC中干燥。 通过LC-MS/MS鉴定蛋白质 LC-MS/MS分析是在配备有纳米喷雾离子源的LTQ Orbitrap发现器质谱仪（Thermo Scientific）上进行的。将标记的肽用200卢斯A期A重悬于（水中的0.1％甲酸）。然后将样品加载到纳米喷雾的锥形毛细管柱/发射器（360x75x15€m，picofrit，picofrit，新目标，沃本，马萨诸塞州，马萨诸塞州）中，在nitrogen压力板中以5分钟的量级（〜5 ul load）在5分钟的班级中，并分别为〜5 ul 5 ul，〜5 ul的额定级别的C18反相树脂（10.5 cm，沃特斯，米尔福德，马萨诸塞州），然后分离〜5 ul B期以〜500 nl/min的流速直接进入质谱仪。使用Turbosequest算法（蛋白质组发现器1.1，Thermo Scientific），针对重组CBH2B-CDNA或CBH2B-yo序列搜索所得数据。将隔离参数设置为允许2 Da的前体离子质量耐受性和0.8 da的片段离子耐受性，具有单异位素质量。允许使用多达两个丢失的内部裂解位点，允许消化的胡椒体，并且允许57.02146 DA，15.9949 DA和136.002 DA的差异修饰分别用于烷基化的半胱氨酸，甲基化的氧化和DTT-LabeL-labeled-Labeled-LabeLed serial或Theonine或Theonine。