不同沉淀剂对酚抽法提取植物蛋白质效果比较分析

酚抽法已经逐渐成为植物蛋白质组研究中通用的蛋白质提取方法,但在各种改进酚抽法中,应用到的沉淀试剂各不相同,致使蛋白沉淀得率、纯度和沉淀时间也各异,本研究以热带植物橡胶树叶片、胶乳和海马齿叶片为研究材料,在过饱和硫酸铵甲醇溶液、醋酸氨甲醇溶液和丙酮溶液沉淀剂下对比蛋白提取的效果,通过单向电泳检测、胶图质量分析、质谱鉴定、蛋白沉淀时间和得率比较,发现3种沉淀剂提取的蛋白样品1-DE图谱显示的蛋白条带数量均较多,但醋酸铵沉淀法提取蛋白图谱有条纹不清晰现象。得出结论：丙酮沉淀法对橡胶树叶片和胶乳蛋白质的提取率较高,硫酸铵沉淀法对海马齿叶片蛋白质提取率较高。过饱和硫酸铵甲醇溶液和丙酮沉淀剂提取的蛋白质质量较高,所得蛋白图谱背景清晰,质谱鉴定蛋白质信息量大。研究结果可优化提取高质量植物蛋白质的方法,并有望为顽拗类植物蛋白质组学研究提供参考。

Comparative Analysis of the Effects of Different Precipitation Agents on Extraction of Plant Protein by Phenol Extract

The borax/PVPP/phenol (BPP) protocol is a widespread protein extraction method in plant proteome research. However, BPP extraction of plant protein, combined with different precipitation agents, the precipitation time, yield and purity of extracted protein is strikingly different. In this study, Sesuvium portulacastrum and Para-rubber tree leaves, and rubber latex were used as the research materials. The plant protein was extracted and analyzed with precipitating agents of supersaturated potassium ammonium sulfate alcohol solution, ammonium acetate methanol solution and acetone solution. Using the methods of SDS-PAGE, gel electrophoresis image quality, mass spectrum identification, settling duration and comparative analysis of protein yield, it was showed that the protein bands extracted by the ammonium acetate precipitation method were not evident. Hevea leaves and latex showed a higher yield of protein in acetone solution. S. portulacastrum leaves had a higher yield of protein in supersaturated potassium ammonium sulfate alcohol solution. The clear background of gel electrophoresis figures was found for the supersaturated potassium ammonium sulfate ethanol solution and acetone solution. The comprehensive protein sequence information identified by mass spectrometry had a higher quality of precipitated protein. The research results can optimize the method of extracting high-quality plant protein, and it will be expected to provide references for recalcitrant plant taxa proteomics research in the future.