PROTEIN IDENTIFICATION AND O-GLYCOSYLATION SITE MAPPING

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： B-消除和Michael加成(BEMAD)用于O-糖基化位点定位 用5 mM DTT在55℃下还原100微克CBH_2b-c DNA和CBH_2b-Yo 1 h，再用15 mM碘乙酰胺在黑暗中甲基化45分钟。干燥透析后的样品用50 mM碳酸氢铵(NH4HCO3)再悬浮，用5g胰酶在37℃下消化20h，在100℃下失活5min，再用5g Glu-C在25℃下消化20h，然后在SpeedVac中干燥。然后将干燥的多肽去除B，并通过再悬浮在1%三乙胺、0.1%NaOH和10 mM DTT中与DTT进行Michael加成。反应在42℃下孵育3h，用1%三氟乙酸熄灭反应。标记的多肽用反相C18柱净化，用0.1%甲酸、80%乙腈洗脱，然后在SpeedVac中干燥。 LC-MS/MS法鉴定蛋白质 LC-MS/MS分析在装有纳米级离子源的LTQ Orbitrap Discoverer质谱仪(Thermo Science)上进行。标记的多肽用200°L流动相A(0.1%甲酸水)重新悬浮。然后将样品以1,000磅/平方英寸(~5微升负载)的氮气压力炸弹自填充于纳米锥形毛细管柱/发射器(360×75×15？m，PicoFrit，New Objective，Woburn，MA)上，用C18反相树脂(10.5 cm，Waters，Milford，MA)自填充5分钟，然后通过160 min的线性梯度增加流动相B，以~500 nL/min的流速直接进入质谱仪。使用TurboSequest算法(Proteome Discoverer 1.1，Thermo Science)对照重组CBH 2b-cDNA或CBH 2b-Yo序列搜索得到的数据。SEQUEST参数被设置为允许2Da的前体离子质量耐受性和0.8Da的碎片离子耐受性与单同位素质量。被消化的多肽最多有两个缺失的内部切割位点，并且允许烷化半胱氨酸、蛋氨酸氧化和DTT标记的丝氨酸或茶氨酸分别进行57.02146 Da、15.9949 Da和15.9949 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.