二维液相色谱法在羊草地上部总蛋白分离中的应用

The Application of Two-dimensional Liquid Phase Chromatography to Separation of Total Protein of Leymus chinensis Shoot

取8周龄羊草的地上部分,用三氯乙酸-丙酮法沉淀总蛋白,沉淀裂解后将缓冲液置换为起始缓冲液,进行第一维色谱聚焦分离。将第一维分离收集的pH值为8.5至4.0之间的组分分别进行第二维无孔硅胶反相高效液相色谱分离,利用ProteoVue软件获得羊草植株总蛋白pI/UV图谱,即羊草植株总蛋白质表达谱。文中对二维液相色谱法分离羊草蛋白质进行了方法学的研究,在第二维分离中尝试用3种不同的洗脱梯度条件进行分离,优化二维液相色谱分离条件并与传统凝胶双向电泳进行了比较,另外还对二维液相色谱的重现性和准确性进行了检验。实验建立了利用二维液相色谱分离羊草总蛋白的技术方法。

The total proteins of Leymus chinensis were extracted from eight-week-old seedlings grown in the greenhouse by TCA-acetone precipi- tation method. After the lysis buffer was replaced by the start buffer, proteins were fractionated along the first dimension using chromatofocusing （CF） （Fig. 1）. Subsequently the fractions with pI values between 8.5 and 4.0 collected after the first dimension separation were further fractionated by nonporous silica reverse-phase high-performance liquid chromatography （NPS-RP-HPLC, HPRP）. With ProteoVue software the pI/UV map （Fig.2） was generated to show the protein expression profile of Leymus chinensis. Some experiments were tested to optimize the fraction procedure. Three different elution gradients were employed to get the optimal chromatogram （Fig.3）. Comparison ofthe protein expression pattern between 2D-PAGE and 2D-LC indicated that 2D-LC was a powerful tool in protein fraction （Fig.4）. Reproducibility and veracity of the protein patterns （Fig.5 and Fig.6） were confirmed in different injections of the same sample. A method to fractionate the total protein of Leymus chinensis shoot with two-dimensional liquid chromatography （2D-LC） was founded.