胆汁双向电泳差异分析样本纯化制备方法的建立

Establishment of a sample preparation strategy for two-dimensional electrophoresis of human bile

目的:建立一种可靠、高效的胆汁样本纯化制备方法用于胆汁蛋白质组双向电泳差异分析。方法:采用梯度SDS-PAGE凝胶电泳的方法比较丙酮、丙酮:甲醇混合液及丙酮:甲醇:磷酸三丁酯混合液及非离子型脱脂剂(CleanasciteTM HC100)的脱脂效果,微量透析管及微量超滤管的脱盐效果及胶体考马斯亮蓝凝胶染色的灵敏度。采用双向荧光差异凝胶电泳比较本组方法与2D-Clean-up Kit对胆汁样本的纯化处理效果。结果:三种沉淀脱脂方法中以丙酮沉淀法效果最佳;非离子型脱脂剂的脱脂效果理想且蛋白回收率高于前者(52.6%比43.7%)。透析法与超滤法脱盐效果均理想,后者耗时短且可以实现蛋白浓缩。胶体考马斯亮蓝染色的灵敏度为10ng。本组方法及2D-Clean-up Kit纯化处理后的胆汁样本均可获得理想的双向电泳图谱,二者分别分离到854和615个蛋白质点。结论:本研究建立了可靠、高效的胆汁样本纯化制备方案,经其处理后的胆汁样本能够满足凝胶双向电泳分析对样本的要求。

Objective To establish a reliable and effective method for bile sample preparation which suitable for two-dimensional electrophoresis. Methods The gradient SDS-PAGE was adopted to compare the delipidation effect of mixture of acetone and methanol, mixture of acetone, methanol and TBP, acetone and non-ionic degreasant （CleanasciteTM HC100）; the desahing effect of dialysis or uhrafiltration; the sensitivity of colloid Comassie brilliant blue staining. And then 2D-GIGE was applied to compare efficiency of our established method and 2-D Clean-up kit in dealing with bile sample. Results It was revealed that acetone had the best effect, while non-ionic degreasant has the equal degreasing effect but higher rate ofprotein recovery compared to acetone（52.6% vs 43.7%）. Bile desalting was realized by dialysis or ultrafiltration device, while the latter one is less time consuming and can complete protein concentration. The highest sen- sitivity of colloid Comassie brilliant blue staining is 10 ng. There are 854 and 615 protein spots being separated in the two-dimensional electrophoresis map by dealing with the same sample using our established method or 2-D Clean-up kit respectively. Conclusions Our research established a reliable and efficient preparation method for purification of bile sample, thus the treated bile sample can satisfy the requirement of biliary two-dimensional electrophoresis.