用于蛋白质组分析的两种马铃薯块茎蛋白质提取方法的比较

Comparison of two extraction methods of potato tuber protein based on proteomic analysis

【目的】进行两种提取方法下马铃薯块茎蛋白质组结果差异分析,并确定最佳马铃薯块茎蛋白质提取方法.【方法】采用酚提取法和三氯乙酸/丙酮沉淀与乙酸铵/甲醇沉淀相结合的两步沉淀法提取马铃薯块茎蛋白质,进行2-DE分离,对差异表达蛋白质进行MALDI-TOF-MS质谱鉴定.【结果】两步沉淀法提取的马铃薯块茎蛋白质较酚提取法获得蛋白质纯度和浓度及蛋白质点数目均明显增加,双向电泳凝胶图谱分辨率较高.质谱鉴定结果表明,两步沉淀法中鉴定的差异表达蛋白质丰度均显著高于酚提取法,并鉴定到一些新出现的蛋白质包括假定的线粒体NAD依赖的苹果酸脱氢酶、酸性磷酸酶类似物和蛋白酶抑制剂II前体类型B.【结论】提取马铃薯块茎蛋白质时两步沉淀法优于酚提取法.

【Objective】 To analyze the difference potato tuber proteomic under two extraction methods,as well as the optimal extraction method of potato tuber protein.【Method】 Potato tuber protein was extracted with phenol extraction method and two-step precipitation method,and then analyzed using two-dimension gel electrophoresis(2-DE),respectively.The differentially expressed proteins were identified by MALDI-TOF/TOF mass spectrometry.【Result】 The purity and concentration of protein extracted with two-step precipitation method was significantly high than that of phenol extraction method,and the number of protein spots and the resolution of 2-DE gel maps were also increased.Mass spectrometry identification results indicated that compared with phenol extraction method,the abundance of protein extracted with two-steps precipitation extraction method was significantly enhanced.And some new proteins were only identified in two-steps precipitation extraction method,including putative mitochondrial NAD-dependent malate dehydrogenase,acid phosphatase-like and proteinase inhibitor II precursor type B.【Conclusion】 Potato tuber protein extracted with two-step precipitation method is better than phenol extraction method.