Establishment and optimization of a two-dimensional gel electrophoresis system for Bactrocera dorsalis (Hendel)larvae crude protein

Author of the article:BAI Rong-Rong1, 2** JIN Tao2 JIN Qi-An2 LIN Yu-Ying2 WEN Hai-Bo2PENG Zheng-Qiang2***

Author's Workplace：1. Environment and Plant Protection Department, Hainan University, Haikou 570228, China; 2. Environment and Plant Protection Key Laboratory of Monitoring and Control of Tropical Agricultural and Forest Invasive Alien Pests, Ministry of Agriculture，Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China

Key Words：Bactrocera dorsalis (Hendel), crude protein extraction methods, two-dimensional gel electrophoresis

Abstract： [Objectives]  To establish and optimize 2-DE profiles of crude proteins from the 3th instar larvae of Bactrocera dorsalis (Hendel). [Methods]  The effectiveness of the BPP protocol and three TCA/acetone precipitation procedures (TCA/acetone A: ground sample with lysis buffer directly, TCA/acetone B: extraction buffer of 10% TCA in acetone containing 40 mmol/LDTT, TCA/acetone C; extraction buffer of 10% TCA in acetone containing 0.07% β-mercaptoethanol), were assessed with respect to crude protein extraction from 3th instar larvae of Bactrocera dorsalis (Hendel). Linear gradient 13 and 24 cm IPG strips with pH 4-7 were used to separate the crude protein. The separation gels were visualized by coomassie brilliant blue and silver staining, respectively. Mass spectra of the specific proteins obtained by the BPP method were read on a 5800 MALDI-TOF-TOF MS/MS mass spectrometer and the database of Metazoa (Animals) and Drosophila (Fruit flies) was indexed for matches. [Results]  The TCA/acetone C precipitation procedure produced the best resolved protein spots among the three TCA/acetone methods, but the BPP method gave the best distinction of proteins on 2-DE gels. Coomassie brilliant blue staining and silver staining showed similar resolutions. The average protein yield of 24 cm strips was higher than that of 13 cm strips, lots of small proteins were enriched in 24 cm strips. The Metazoa database provided more comprehensive information than that of Drosophila (Fruit flies). [Conclusion]  The BPP protocol revealed the most protein spots with linear gradient 24 cm IPG strips, pH 4-7. Coomassie brilliant blue was both sensitive and compatible with MS. Database retrieval should give priority to the Metazoa.