小鼠血浆去除白蛋白前后的双向凝胶电泳分析

An approach to remove albumin for the proteomic analysis of biomarkers in mouse plasma

目的 探索应用DEAE CibacronBlue 3GA去除小鼠血浆白蛋白的条件。方法 在DEAE CibacronBlue 3GA与小鼠血浆结合的不同时间点取样 ,SDS PAGE及蛋白质定量检测去除白蛋白的效果 ,对最佳结合时间点所取的血浆行双向凝胶电泳 ;软件分析去除白蛋白前后双向电泳显示的蛋白质斑点变化。结果 DEAE CibacronBlue 3GA与小鼠血浆结合 40min时 ,小鼠血浆中80 %的白蛋白被去除 ,双向电泳显示的蛋白质点由处理前的 (2 11± 19)个增加到 (3 92± 11)个 ;其中蛋白表达量增加 3、5、10、2 0倍的斑点数目分别为 13、6、6、5。结论 DEAE CibacronBlue 3GA可以有效去除小鼠血浆中的白蛋白 ,该方法可以应用于实验外科领域小鼠血浆蛋白质组的研究。

Objective To reduce the interference fromthe plasma abundant proteins in two-dimensional gel electrophoresis (2-DE),a sample preparation to remove albumin and to enrich low abundant proteins in plasma was developed.Methods Plasma samples of mouse were treated with DEAE-Cibacron Blue 3GA and then subjected to 2-DE.Protein spots were visualized by silver staining and further analyzed by Phoretix 2D v2003.02.Results Comparison of the 2DE images for the mouse plasma treated with and without the affinity resin revealed that albumin was removed almost 80% and the stained spots were significantly increased from 211±19 to 392±11 due to the treatment.DEAE-Cibacron Blue 3GA treatment for 30 min resulted in enhanced visualization of 13 protein spots by 3-fold,6 by 5-fold,6 by 10-fold and 5 by 20-fold.Conclusion The plasma preparation using the affinity resin is an efficient approach to remove albumin and results in high quality of 2DE images for the analysis of differential proteomics in plasma.