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首页> 外文期刊>Journal of proteome research >Large-Scale Quantitative Proteomic Study of PUMA-lnduced Apoptosis Using Two-Dimensional Liquid Chrpmatography-Mass Spectrometry Coupled with Amino Acid-Coded Mass Tagging
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Large-Scale Quantitative Proteomic Study of PUMA-lnduced Apoptosis Using Two-Dimensional Liquid Chrpmatography-Mass Spectrometry Coupled with Amino Acid-Coded Mass Tagging

机译:二维液相色谱-质谱联用氨基酸编码的质谱标记技术对PUMA诱导的细胞凋亡进行大规模定量蛋白质组学研究

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By coupling two-dimensional liquid chromatography-tandem mass spectrometry(2D-LC-MS/MS)with amino acid-coded mass tagging(AACT),we have greatly increased the analytical throughput and sequence coverage of MS-based methods for proteome-wide quantitation.The dynamic range and reproducibility of this 2D-LC-AACT quantitative approach were evaluated by profiling the mixtures with different ratios of E.coli cells grown in either regular or AACT medium.A SQL-based high thoughput MASCOT data analysis tool was developed for proteomic data sorting and mining.We investigated the early stage of apoptosis by inducing the p53 upregulated modulator of apoptosis(PUMA)through the analyses of the relative ratios of the pairwise isotope signals that were originated from the control and labeled PUMA-induced cells.In 20-hour 2D-LC-MS/MS run,480 proteins were conclusively identified,and more than half of them were quantified.A noteworthy change in the quantitative profile was that histones and a ubiquitin conjugate protein UBC9,which are involved in DNA double-strand break(DSB)repair were significantly down-regulated in the PUMA-overexpressing apoptotic cells,suggesting the detection of DSB in the apoptotic process.The quantitative profiling efficiency of this approach was compared with the gel-based quantitative analysis scheme.
机译:通过将二维液相色谱-串联质谱(2D-LC-MS / MS)与氨基酸编码的质谱标签(AACT)结合使用,我们大大提高了基于蛋白质组的全质谱方法的分析通量和序列覆盖率通过对常规或AACT培养基中生长的不同比例的大肠杆菌细胞进行分析,评估了该二维LC-AACT定量方法的动态范围和可重复性。开发了基于SQL的高通量MASCOT数据分析工具我们通过分析来自对照和标记的PUMA诱导细胞的成对同位素信号的相对比率,通过诱导p53上调凋亡调节剂(PUMA)来研究凋亡的早期阶段。在20小时的2D-LC-MS / MS运行中,最终鉴定出480种蛋白质,其中一半以上被定量。定量配置中值得注意的变化是组蛋白和泛素c在过表达PUMA的凋亡细胞中,参与DNA双链断裂(DSB)修复的UUB9结合蛋白UBC9明显下调,建议在凋亡过程中检测DSB。将该方法的定量分析效率与基于凝胶的定量分析方案。

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