Integrative proteomics of primary hepatocytes during dedifferentiation

To understand the relationship between protein expression and mRNA translation during primary hepatocytes dedifferentiation, we have employed transcriptome microarrayas a discovery platform. Rat primary hepatocytes were isolated by the method of two-step enzymes perfusion and then cultured on mono-layer in vitro. Samples at 0h( just after perfusion, before planking) , 6h, 12h ,24h and 48h were collected. Integrative analysis of transcriptome and whole cell proteomics (WCP) leaded us to realize the poor correlation of them. This discovery made us realize that targeting mRNA was far from enough in illustrating this process. It would provide new insights from the aspects of post-translational modifications(PTMs).Post-translational modifications play important role in numorous biological and pathological process, but a few reports are related to primary hepatocytes dedifferentiation process, and there is still no integrative proteomics analysis in this field yet. In this study, we perform ubiquitinome phosphorylated proteome, whole cell proteome and transcriptome simultaneously during the five different time points of dedifferentiation in vitro quantified over 6000 modified sites mapping to over 2000 proteins. And comprehensive analysis of these datasets provides novel insight in this field.为探究原代肝细胞去分化过程中蛋白质表达与mRNA翻译的关联，我们选用转录组微阵列（transcriptome microarray）作为发现级研究平台。我们采用两步酶灌流法分离大鼠原代肝细胞，并将其体外单层培养。分别在灌流结束后、铺板前的0小时，以及培养6小时、12小时、24小时和48小时收集样本。通过对转录组与全细胞蛋白质组（whole cell proteomics, WCP）开展整合分析，我们发现二者相关性较弱。这一发现提示，仅靶向mRNA远不足以阐明该去分化过程，后续可从翻译后修饰（post-translational modifications, PTMs）角度为该领域提供全新研究视角。翻译后修饰在众多生理与病理过程中发挥关键作用，但目前针对原代肝细胞去分化过程的相关研究较少，且该领域尚未有整合蛋白质组学分析的相关报道。本研究在体外原代肝细胞去分化的五个不同时间点，同时开展泛素化修饰组、磷酸化蛋白质组、全细胞蛋白质组与转录组分析，共定量到超过6000个修饰位点，对应2000余种蛋白质。对上述多组学数据集的综合分析将为该领域提供全新的研究见解。