双向凝胶电泳脑脊液蛋白质提取方法的比较

Comparison of the extracting methods of cerebrospinal fluid proteins using two-dimensional polyacrylamide gel electrophoresis

目的对3种常用的双向凝胶电泳脑脊液蛋白质提取方法进行比较,优选脑脊液蛋白质提取方法。方法收集新诊断尚未经药物治疗的16例帕金森病患者和7例头痛患者的脑脊液,分别以3种不同的方法提取脑脊液中的蛋白质。①透析法:用PluseOne微透析试剂盒处理脑脊液样品。②丙酮沉淀法:以冷丙酮溶液处理脑脊液样品,丙酮的终浓度为80%。③三氯醋酸(TCA)/丙酮沉淀法:以4倍体积的10%TCA/丙酮溶液处理脑脊液样品,TCA的终浓度为8%,丙酮的终浓度为80%。取沉淀物进行双向凝胶电泳,用银染法对电泳后的凝胶进行染色,比较经3种不同方法提取的脑脊液蛋白质的双向凝胶电泳图谱。结果经透析法处理样品的电泳图谱显示的蛋白点较清晰、较圆,条纹较少,但所见几乎都是高丰度蛋白,低丰度蛋白很难显现;经丙酮沉淀法处理样品的电泳图谱显示横竖条纹均较多,大部分蛋白堆积在凝胶的上部,下面的点也显示不清晰,蛋白点出现横向漂移;经TCA/丙酮沉淀法处理样品的电泳图谱显示横条纹相对较少,低丰度蛋白能较清晰显现,样品中所含高丰度蛋白明显少于以上两种方法处理的样品。结论以TCA/丙酮沉淀法提取蛋白质进行双向凝胶电泳时聚焦效果较好,而且同时去除了大部分白蛋白,使低丰度蛋白能很好地显现出来,优于丙酮沉淀法和透析法。

Objective To find an optimal extracting method of cerebrospinal fluid proteins by comparing three methods using two-dimensional polyacrylamide gel electrophoresis(2-DE). Methods Cerebrospinal fluid was collected by lumbar puncture from 16 patients with Parkinson's disease and 7 patients with headaches,who had not been treated. The cerebrospinal fluid proteins in the samples were precipitated with dialyzing,acetone,or Trichloroacetic acid(TCA) /acetone methods. ①Dialysis method:The cerebrospinal fluid samples were processed with the Pluse One microdialysis kit. ②Acetone precipitation method:Cerebrospinal fluid proteins were precipitated with cold acetone,and the final concentration of acetone was 80%. ③TCA /acetone precipitation method:Cerebrospinal fluid proteins were extracted with 10%TCA/ acetone,the volume of TCA/ acetone was 4 times of that of the cerebrospinal fluid samples. In other words,the finalconcentration of TCA was 8%,and the final concentration of acetone was 80%. Proteins precipitated from the cerebrospinal fluid were run by immobilized pH gradient isoelectic focusing(IPG-IEF),and then by vertical flat sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE),the protein spots in the gels were silver-stained and observed;and then the gels images were collected and compared. Results By dialysis method,the protein spots were clear and round with fewer streaks on the images,however,almost all the proteins that could be seen were high-abundance proteins,few low-abundance proteins could be observed. By acetone precipitation method,more horizontal and vertical streaks were shown on the images,most of the proteins were piled up on the upper part of the gels,the spots on the lower part were unclear;moreover,the protein spots were drifted transversally. By the TCA/acetone precipitation method,horizontal and vertical streaks were relatively fewer,the protein spots were separated better,low-abundance proteins were shown more clearly,and high-abundance proteins in the image were obviously less than those in the images of proteins precipitated with dialysis or acetone. Conclusions The protein precipitation method with 10% TCA/acetone could remove salt better,increase the focus effect,and remove most of the albumin,so that the low-abundant proteins can be shown more clearly in 2-DE images. Thus,we consider that the TCA/ acetone precipitation method is superior to the dialyzing and acetone methods.