An evaluation of protein extraction methods suitable for Proteomic Analysis of Plutella xylostella

Abstract

Insects are major pests of agricultural that cause damage and economic loss of crops. They induce high loss in crop yield by directly feeding on plants and indirectly transmitting various virus diseases. The Diamondback moth, Plutella Xylostella is one of the most destructive insect pests of Cruciferous crops throughout the world. The extensive use of insecticides for controlling this pest has resulting the emergence of resistance and causing major problem for the farmers. In order to effectively manage resistance and for the crop protection, it is vital to understand the biochemical and physiological adaptations of P. Xylosytella against insecticides. Insect proteomics has become a rapidly evolving field of research, wherein efforts are continuing for profiling and analyze the Proteome content in insects at the organelle level and individual tissue. By using Proteomics approach, P. Xylostella proteins interacting with insecticides can be identified, and their modification in resistant strains can be characterized. A critical step in proteomics is the protein preparation and isolation. The main objective of this study is to evaluate protein extraction method that suitable for larvae and adult tissues of P. xylostella. Five extraction methods were evaluated in terms of protein concentration and resolving patterns of one dimensional electrophoresis (1-DE). Proteins from larvae and adult tissues of P. xylostella were extracted using three modified trichloroacetic acid (TCA)/acetone, lysis buffer and phosphate buffer methods. Among the five methods compared, modified TCA/acetone with P-mercaptoethanol produced the highest protein yields. In SDS-PAGE, the extracts from modified TCA/acetone/13-mercaptoethanol and TCA/acetone/dithiothreitol (DTT) showed a clear protein profiles with well-defined bands from 17 kDa to 245 kDa. These extraction methods therefore can be used in further work such as 2-dimensional electrophoresis (2-DE) and mass spectrometry (MS) in order to analyze Proteome in P. Xylostella. To our, knowledge, this is the first report on protein extraction methods for both larvae and adult tissues of P. Xylostella.