乙腈、乙醇、色谱柱去除血清高丰度蛋白方法的比较

Comparison of acetonitrile,ethanol and chromatographic column to eliminate high-abundance proteins in human serum

目的比较乙腈沉淀法、乙醇沉淀法和多重亲和去除色谱柱Human 14法去除人血清中高丰度蛋白的效果。方法应用乙腈沉淀法、乙醇沉淀法和多重亲和去除色谱柱Human 14法对血清样品进行处理,Bis-Tris Mini Gels电泳和双向凝胶电泳检测去除高丰度蛋白的效果。结果从一维胶电泳图灰度值分析,血清经过乙腈法、色谱柱法、乙醇法处理后,白蛋白条带灰度值由原血清的19分别变成157.2,40.8和8.2;从2-DE电泳图分析看出,多重亲和去除色谱柱Human 14法处理后的蛋白点数显著增多,比原血清增加了137个;乙腈法和乙醇法处理后虽然蛋白点减少了,却有低丰度蛋白质点显现出来。结论乙腈沉淀法能在去除血清中高丰度蛋白的同时收获更多的10kD以下的小分子量蛋白;乙醇沉淀法虽然能去除部分高丰度蛋白,但小分子量蛋白的收获量较少;而多重亲和去除色谱柱Human 14法不仅能有效地去除血清中高丰度蛋白的干扰,且保留了大量的小分子量蛋白。

Objective To compare the effects of acetonitrile precipitation,ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum.Methods Elimination of serum high-abundance proteins performed with acetonitrile precipitation,ethanol precipitation and multiple affinity chromatography column Human 14 removal.Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect.Results Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method,multiple affinity chromatography column Human 14 removal method and ethanol method,the grey value of albumin changed into 157.2,40.8 and 8.2 respectively from the original value of 19.2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method,the protein points noticeable increased by 137compared to the original serum.After processed by acetonitrile method and ethanol method,the protein point reduced,but the low abundance protein point emerged.Conclusions The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight,ethanol precipitation could eliminate part of high abundance proteins in serum,but low abundance proteins less harvested,and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins,and keep a large number of low abundance proteins.