伴刀豆球蛋白A诱导肝细胞损伤的磷酸化蛋白质组学研究

Phosphoproteomic analysis of concanavalin A-induced hepatocytes injury

目的·研究伴刀豆球蛋白A(concanavalin A,ConA)诱导肝细胞损伤过程中全细胞蛋白质的磷酸化修饰水平的变化规律。方法·将小鼠肝细胞株AML12与ConA(10μg/mL)共培养,诱导肝细胞损伤模型,12 h后提取细胞的总蛋白,经消化酶解后富集肽段,并使用高效液相-质谱联用分析仪进行检测。采用非标定量法对磷酸化位点和肽段进行鉴定与相对定量分析。筛选出单位点磷酸化修饰水平差异达到2倍以上的蛋白进行生物学功能富集分析和蛋白互作调控网络分析。结果·在ConA诱导的AML12细胞损伤模型中,共鉴定到磷酸化修饰肽段11200条,可定位定量的肽段2685条。其中差异性调控的磷酸化修饰肽段82条,对应77个蛋白。这77个磷酸化蛋白主要与蛋白质结合、酶活动等相关,且主要通路与细胞凋亡以及RAS激酶通路相关。结论·AML12细胞在ConA诱导的肝细胞损伤过程中发生了磷酸化修饰变化,差异磷酸化修饰的蛋白可能与肝细胞凋亡相关。

Objective·To study the changes of phosphorylation level of whole-cell proteins during the process of concanavalin A(ConA)-induced hepatocyte injury.Methods·Mouse hepatocytes of AML12 cells and ConA(10μg/mL)were co-cultured to induce a hepatocyte injury model.After 12 h,the total proteins were extracted and digested into peptides.The peptides were enriched by metal oxide affinity chromatography and detected by using high performance liquid chromatography-mass spectrometer.Identification and relative quantitative analysis of phosphorylation sites and peptides were performed using the Label free method.Proteins with a difference of more than two times of phosphorylation level of each site were screened.And the biological function enrichment analysis and protein interaction regulation network analysis were performed.Results·In the ConA-induced AML12 cell injury model,a total of 11200 phosphorylated modified peptides and 2685 localizable and quantifiable peptides were identified.Among them,there were 82 differentially regulated phosphorylated modified peptides,corresponding to 77 proteins.These phosphorylated proteins were mainly related to protein binding and enzyme activities.And the main pathways were related to apoptosis and RAS kinase pathways.Conclusion·AML12 cells undergo phosphorylation level change in ConA-induced liver cell injury and these differentially phosphorylated proteins may be related to liver cell apoptosis.