不同转移潜能的肝癌细胞系定量磷酸化蛋白质组学研究

Quantitative phosphoproteomics study of hepatocellular carcinoma cell lines with different metastatic potential

目的研究具有不同侵袭转移能力的肝癌细胞系的磷酸化蛋白质组的动态变化。方法首先用含有重稳定同位素标记的精氨酸和赖氨酸的培养基对MHCC97-L进行重稳定同位素标记氨基酸的细胞培养(SILAC);用含有轻稳定同位素标记的精氨酸和赖氨酸的培养基对MHCC97-H进行SILAC标记。将完成标记的两种肝癌细胞系的蛋白质按照1∶1混合,并用trypsin进行溶液消化;对消化得到的肽段进行除盐,并通过固相金属离子亲和色谱(IMAC)的方法进行磷酸化肽段的富集。磷酸化肽段通过质谱进行检测,并通过MaxQuant进行鉴定和定量。最后通过Z检验进行统计学分析,并对差异调控的磷酸化肽对应的蛋白质进行基因本体(GO)分析和STRING网络分析。结果最终鉴定了918个磷酸化肽段,其中806个磷酸化肽段的磷酸化位点可以定位和定量。通过Z检验,共得到了91个磷酸化修饰水平发生变化的肽段,对应25个磷酸化蛋白质。通过对这25个蛋白质进行GO分析发现,细胞骨架重塑过程中的蛋白质在MHCC97-L和MHCC97-H中磷酸化修饰形式严重失调。通过STRING分析发现,NES和PRKCA处于网络的重要节点位置。结论细胞骨架重塑这一生物学过程与肝癌侵袭转移能力密切相关;参与细胞骨架重塑的NES蛋白和参与MAPK信号通路和黏合斑信号通路的PRKCA蛋白磷酸化修饰水平的变化是影响低转移潜能MHCC97-L细胞向高转移潜能细胞MHCC97-H变化的重要调控机制。

Objective To study the phosphoproteome dynamic change of hepatocellular carcinoma （HCC） cell lines with different invasion and metastasis potential. Methods MHCC97-L was cultured in medium containing heavy isotope labeled lysine and arginine by stable isotope labeling with amino acids in cell culture（SILAC）. And MHCC97-H was cultured in medium containing light isotope labeled lysine and arginine. Proteins of completely la- beled MHCC97-L and MHCC97-H were mixed at 1 ： 1 ratio and digested by trypsin. Digested peptides were desalted and the phosphopeptides were enriched by immobilized metal ion affinity chromatography（IMAC） and analyzed by LC-MS/MS （liquid chromatography-mass spectrometry）. The MS data were processed using the MaxQuant soft- ware. Quantified phosphopeptides were filtered by Z-test for altered phosphopeptides. The proteins were analyzed by gene ontology（GO） biological processes and STRING protein-protein interaction. Results 918 phosphopeptides were identified, 806 of which could be localized and quantified. By means of Z-test, 91 phosphopeptides, which were corresponding to 25 phosphoproteins, altered between MHCC97-L and MHCC97-H. According to GO analy- sis, cytoskeleton organization seriously perturbed between these two cell lines. STRING protein-protein interaction indicated that NES and PRKCA were two key proteins. Conclusion Cytoskeleton organization dysfunction is close- ly related to invasion and metastasis between these two cell lines. Further analysis indicates that phosphorylation of NES and PRKCA, which is involved in cytoskeleton organization and MAPK signaling pathway separately, plays an important role during metastasis of HCC.