小麦叶片蛋白质组的2D-LC分离及Nano LC-MS/MS分析

 【目的】二维液相色谱（2D-LC）与双向电泳（2-DE）技术具有互补性，本文旨在探讨利用2D-LC和纳升级液相色谱串联质谱（Nano LC-MS/MS）技术分离鉴定小麦叶片蛋白质组的新方法。【方法】小麦叶片蛋白经提取、脱盐后，进行第一维阴离子交换分离;收集洗脱组分进行SDS-PAGE分析，并对非高丰度蛋白组分进行第二维反相液相色谱分离;随机选择含较低吸收峰的部分组分经胰蛋白酶水解后进行Nano LC-MS/MS 分析;将串联质谱数据通过MASCOT搜索NCBInr和EST数据库，并对二级质谱获得的蛋白序列进行MS BLAST分析。【结果】经第一维阴离子交换色谱分离，收集得到15个组分，其中第15组分包含小麦叶片丰度最高的蛋白——1,5-二磷酸核酮糖羧化酶/加氧酶（RuBisCO）;其余14个组分经反相液相色谱分离，共收集1 551个组分，获得1 867个色谱峰;随机对其中6个含较低吸收峰的收集组分酶解和Nano LC-MS/MS 检测，9种蛋白得到鉴定。【结论】结果初步表明，利用2D-LC和Nano LC-MS/MS技术可有效地分离和鉴定小麦叶片蛋白质组，为更深入全面地研究小麦叶片蛋白质组奠定基础。

Separation and Analysis of Wheat Leaf Proteome by Combination of 2D-LC and Nano LC-MS/MS

【Objective】 Two-dimensional liquid chromatography (2D-LC) can be used as a complementary approach to protein separation with two-dimensional gel electrophoresis (2-DE). The purpose of the study is to establish a protocol to separate and identify wheat leaf proteome by combination of 2D-LC and Nano LC-MS/MS. 【Method】 The soluble protein extracted from wheat leaf were desalted by gel filter chromatography and separated by strong anion exchange chromatography (SAX) in the first dimension. The elution fractions were subjected to SDS-PAGE and the fractions without the most abundant protein (s) were further separated by reversed phase liquid chromatography (RPLC) in the second dimension. To test the effectiveness of the separation method, some of the 2D-LC fractions including lower intensity peaks were digested and analyzed by Nano LC-MS/MS. The MS/MS data were used to search against NCBInr and EST database using MASCOT search engine. Meanwhile, de novo sequencing was performed manually and the data were used to search against nrdb95 database using MS BLAST search engine. 【Result】 A total of 15 collections were obtained through the first dimensional separation and the most abundant protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in plant leaf, could only be observed in the 15th collection. After the second dimensional separation, 1551 collections were collected with 1867 resolved protein peaks from the other 14 fractions without RuBisCO. Nine proteins were identified from six 2D-LC collections with lower intensity peaks selected randomly. 【Conclusion】 Based on the experiment setup and results, it is tentatively concluded that the combination of 2D-LC and Nano LC-MS/MS could be an effective method in future wheat leaf proteomics analysis.