Abstract:

【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.

Key words: proteomics, wheat leaf, 2D-LC, Nano LC-MS/MS