人胆汁双向凝胶电泳分离技术的建立与优化

背景：双向凝胶电泳是目前最好的蛋白质分离方法，然而胆汁样品中的盐、脂和蛋白酶对双向凝胶电泳干扰较大，影响电泳图谱的重复性。目的：通过对胆汁样品双向凝胶电泳的几个关键环节的分析，建立起一种适合于有效分离胆汁蛋白质的双向凝胶电泳分离方法。设计、时间及地点：胆汁双向凝胶电泳图谱对照实验，于2008-03/11在重庆市神经病学重点实验室完成。材料：胆汁标本取自于重庆医科大学附属第一医院肝胆外科，于肝移植过程中从供体中取得。方法：将标本分别按3种不同方法进行处理：①2D-Clean-up试剂盒处理胆汁标本：用裂解液，7 mol/L尿素，2 mol/L硫脲，质量浓度为0.4 g/L的过硫酸铵，65 mmol/L二硫苏糖醇，质量浓度为0.2 g/L的载体两性电解质溶解沉淀过夜。②在胆汁标本中加入4倍体积的预冷丙酮，用预冷的丙酮洗涤沉淀，然后用真空冻干机抽干，用裂解液溶解沉淀过夜。③在胆汁标本中加入4倍体积的预冷三氯乙酸/丙酮混合液，用预冷的丙酮洗涤沉淀，然后用真空冻干机抽干，用裂解液溶解沉淀过夜。经过一维等电聚焦和二维SDS-PAGE电泳分离人胆汁蛋白质组。将切取的蛋白质点进行脱色，酶解，萃取，冷冻抽干后，进行质谱鉴定。主要观察指标：胆汁双向电泳图谱上蛋白质点数目，位置及重复性的比较。结果：3种方法均能得到理想的一向电泳等电聚焦，与单纯的丙酮沉淀法相比较，但三氯乙酸/丙酮沉淀法损失了较多的中小分子量的蛋白质，因此不适合于胆汁标本的处理。与2D-Cleanup试剂盒处理法相比，丙酮沉淀法纯化胆汁内盐、脂较丰富，去除干扰成分，具有较好的效果，具有成本低廉、操作简便等优势。结论：通过优化胆汁蛋白质提取、上样量和双向电泳重复性检测等步骤，建立了人胆汁双向凝胶电泳的方法，丙酮沉淀与双向凝胶电泳技术的结合是获取人胆汁蛋白质组的一种有效方法。

Construction and optimization of a separation technique of human bile using two-dimensional gel electrophoresis

BACKGROUND: At present, two-dimensional gel electrophoresis is the best method for protein separation. However, electrophoretogram, which can be interfered with salt, lipid or protease in bile sample, is poor in repetitiveness.OBJECTIVE: To establish and optimize the two-dimensional gel electrophoresis separation of proteome by analyzing key link human bile.DESIGN, TIME AND SETTING: The comparison experiment of two-dimensional gel electrophoresis of bile was performed at the Chongqing Key Laboratory of Neurology from March to November in 2008.MATERIALS: The bile was obtained from donator during liver transplantation at the Department of Hepatobiliary Surgery, First Affiliated Hospital, Chongqing Medical University.METHODS: The bile was treated with 3 different methods, including the 2D-Clean-up kit group: the bile sample was dissolved with lysis buffer (contains: 7 mol/L carbamide, 2 mol/L thiourea, ammonium persulfate with mass concentration of 0.4 g/L, 65 mmol/L dithiothreitol, carrier ampholytes with mass concentration of 0.2 g/L). The acetone precipitation group: bile sample was washed with 4 times volume of precooled acetone, dried by vacuum freeze dryer and dissolved with lysis buffer. The trichloracetic acid/acetone precipitation group: 4 times volume of mixture of trichloracetic acid/acetone was added in the bile sample, washed with 4 times volume of precooled acetone, dried by vacuum freeze dryer and dissolved with lysis buffer. The proteome in human bile were separated by isoelectric focusing on first dimension and SDS electrophoresis on second dimension. Protein spots were identified by mass spectrometry identification.MAIN OUTCOME MEASURES: Comparison of number, position and reproducibility of the protein spots on the two-dimensional gel electrophoresis of bile.RESULTS: Isoelectric focusing on first dimension could be obtained by each method. Compared with the acetone precipitation group, majority of protein with medium or small molecule was lost in the trichloracetic acid/acetone precipitation group, which was incompetence for the treatment of bile. The acetone precipitation group had the superiority than the 2D-Clean-up kit group in cost, operation or treating results.CONCLUSION: By optimizing the procedure of protein extraction, sample volume as well as detected repetition, two-dimensional gel electrophoresis can be constructed, which is a suitable approach to study the proteome in human bile combined with acetone-precipitation.