Proteomics of HTR8 cells with bile acids and EBSS treatment

HTR8 cells maintained in Earle's balanced salt solution (EBSS) and treated with 100 μM chenodeoxycholic acid (CDCA) or 100 μM deoxycholic acid (DCA) for 18 h were subjected to proteomic analysis After treatment with bile acids (CDCA or DCA) and EBSS, HTR8 cells (two duplicates in each group) were harvested and lysed. The protein lysates of HTR8 cells were digested with trypsin (Promega, Madison, WA, USA). The resulting peptide in each sample was then labeled using a Tandem Mass Tags (TMT) Labeling kit (ThermoFisher Scientific) following the manufacturer’s instructions and fractionated by reverse-phase chromatography using high-performance liquid chromatography (1260 Infinity II; Agilent, USA). Liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was performed on a mass spectrometer (positive ion mode; Q Exactive Plus; ThermoFisher Scientific) coupled with a nanoflow LC system (Easy-nLC; ThermoFisher Scientific). MS data were acquired and processed using MASCOT (version 2.6; Matrix Science, London, UK) embedded into Proteome Discoverer 2.2. Proteins with fold changes higher than 1.2 or less than 0.83 and p values less than 0.05 were considered as differentially expressed.将维持于Earle平衡盐溶液（EBSS）中的HTR8细胞分别用100 μM鹅脱氧胆酸（CDCA）或100 μM脱氧胆酸（DCA）处理18小时后，开展蛋白质组学分析。经胆汁酸（CDCA或DCA）与EBSS处理后，收集各组HTR8细胞（每组设两个生物学重复）并裂解。将HTR8细胞的蛋白裂解液用胰蛋白酶（Promega，美国威斯康星州麦迪逊市）进行酶解。按照制造商说明书，采用串联质量标签（TMT）标记试剂盒（赛默飞世尔科技）对各样本中的所得肽段进行标记，并通过高效液相色谱（1260 Infinity II；安捷伦，美国）结合反相色谱法完成分级分离。采用耦合纳流液相色谱系统（Easy-nLC；赛默飞世尔科技）的质谱仪（正离子模式；Q Exactive Plus；赛默飞世尔科技）完成液相色谱（LC）-质谱（MS）/质谱分析。采用嵌入Proteome Discoverer 2.2中的MASCOT软件（版本2.6；Matrix Science公司，英国伦敦）采集并处理质谱数据。将倍数变化大于1.2或小于0.83、且p值小于0.05的蛋白认定为差异表达蛋白。