PROTEIN IDENTIFICATION AND O-GLYCOSYLATION SITE MAPPING

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： B-消除后Michael加成（BEMAD）用于O-糖基化位点作图 将100 μ g CBH 2b-cDNA和CBH 2b-YO在55 ℃下用5 mM DTT还原1 h，并在黑暗中用15 mM碘乙酰胺羧酰胺甲基化45 min。将干燥的透析样品重悬于50 mM碳酸氢铵（NH 4 HCO 3）中，并在37 ℃下用5 μ g胰蛋白酶消化20 h。在100 ℃下使蛋白酶失活5分钟后，将样品用5 μ g Glu-C在25 ℃下消化20小时，然后在Speed Vac中干燥。然后将干燥的肽进行B-消除，并通过重悬于1%三乙胺、0.1%NaOH和10 mM DTT中进行与DTT的Michael加成。将反应在42 ° C下孵育3小时，并用1%三氟乙酸淬灭反应。通过反相C18柱净化标记的肽，在0.1%甲酸、80%乙腈中洗脱，并在Speed Vac中干燥。 蛋白质鉴别（LC-MS/MS） 在配备有纳米喷雾离子源的LTQ Orbitrap Discoverer质谱仪（Thermo Scientific）上进行LC-MS/MS分析。用200 μ L移动的A相（0.1%甲酸水溶液）重悬标记的肽。然后将样品加载到纳米喷雾锥形毛细管柱/发射器上。（360 x75 x15英寸）m，PicoFrit，New Objective，Woburn，MA）用C18反相树脂自填充（10.5 cm，沃茨，米尔福德，MA）在氮气压力瓶中于1，000 psi（约5 uL上样量），然后通过160 min线性梯度递增的移动的相B以约500 nL/min的流速直接分离至质谱仪中。使用TurboSequest算法（Proteome Discoverer 1.1，Thermo Scientific）针对重组CBH 2b-cDNA或CBH 2b-YO序列搜索所得数据。SEQUEST参数设置为允许单一同位素质量的2Da前体离子质量容限和0.8Da碎片离子容限。允许消化的肽具有多达两个缺失的内部切割位点，并且允许分别针对烷基化半胱氨酸、甲硫氨酸氧化和DTT标记的丝氨酸或苏氨酸的57.02146 Da、15.9949 Da和136.002 Da的差异修饰。

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.