【摘要】 目的:对人胚胎干细胞H9的(磷酸化)蛋白质组进行鉴定和深入分析,探讨维持人胚胎干细胞干性的核心调控网络。方法:利用新近发表的TAFT磷酸化肽段富集策略和高精度质谱鉴定技术,采用无标定量方法对H9细胞(磷酸化)蛋白进行定量分析;结合MOTIFX、iGPS、IPA等多种生物信息学分析软件,分析H9细胞的磷酸化基序-激酶、转录因子-靶基因调控网络。结果:获得了目前高度覆盖的H9细胞(磷酸化)蛋白质组数据集,鉴定到8674种蛋白质,其中3898种能够发生磷酸化修饰,包括13 676条磷酸化肽段,高可信度(class 1)磷酸化位点有11 870种。与已有的H9细胞磷酸化蛋白质组数据相比,其中2247种磷酸化蛋白质和10 025种磷酸化位点是本研究新鉴定到的。基于基序和激酶预测分析,推导出与干性维持相关的已知转录调节因子的新的磷酸化修饰基序以及调控其发生磷酸化的激酶。结合多能性转录因子对靶基因的调控信息,构建了多能性调控分子磷酸化修饰及其转录调控的核心网络。此外,还对特定位点的磷酸化修饰水平及其对应蛋白的总体丰度水平进行了比较及功能分析。基于各自的百分位数,比较磷酸化修饰水平与其对应蛋白的总体丰度,发现具有高丰度但低磷酸化修饰水平的蛋白倾向于参与物质转换或物质运输等生物过程,而低丰度但高磷酸化水平的蛋白质倾向于参与信息转导或调控过程。结论:提供了人胚胎干细胞H9高覆盖的(磷酸化)蛋白质组数据,这些数据有助于深入了解蛋白质磷酸化修饰在胚胎干细胞干性维持中所发挥的重要作用,为胚胎干细胞基础和应用研究提供了宝贵的数据资源。更多还原

【Abstract】 Objective: Based on the（phospho）proteome dataset of the H9 embryonic stem cells（ESC）, the core regulatory networks for the maintenance of stemness in human ESC（h ESC） were explored. Methods: A newly published strategy named TiO₂ with tandem fractionation（TAFT） and high-resolution mass spectrometry was employed.The（phospho）proteome of H9 cells was analyzed quantitatively using label-free strategy. Combined with various bioinformatic softwares such as MOTIFX, iGPS and IPA, the regulation network of motif-kinase and transcription factor-target genes of H9 cells was analyzed. Results: The largest（phospho）proteome dataset of the H9 ESC to date was obtained, including 8674 unique proteins, in which 3898 were identified as phosphoproteins with 13 676 phosphopeptides and 11 870 high confidence（class 1） phosphosites. Compared with the existing（phospho）proeome data of H9 cells, 11 314 phosphoproteins and 10 025 phosphorylation sites were newly identified in this study.Given to the motif and kinase of the high confidence phosphorylation sites identified here, we further inferred the predominant linear kinase motifs and corresponding kinases to these motifs. Through the analysis about transcription factors（TF） and target genes（TG） regulatory network, the core regulatory network controlling stemness was reconstructed. When comparing the difference between the percentile rank of the intensities of specific phosphosites and the percentile rank of the i Based Absolute Quantification（i BAQ） values of corresponding proteins, it was found that the proteins with high abundance but low phosphorylation levels tend to participate in the processes of material conversion or transportation, whereas the proteins with low abundance but high phosphorylation levels tend to participate in the processes of information transduction or regulation. Conclusion: This study provided a highcoverage of（phospho）proteome of h ESC, which is important to the deep understanding of molecular mechanism of stemness maintenance in h ESC, and is valuable for basic and applied research of stem cell.更多还原