Methodology for glutathione S-transferase purification and localization in two-dimensional gel electrophoresis performed on the pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae)

Abstract

Glutathione S-transferases (GSTs) are one of the major detoxification enzymes involved in insecticide resistance. In this study, optimized methodology for GSTs purification and localization in two-dimensional gel electrophoresis is shown on the pollen beetle. Specifically, the GST proteins were purified using GSTrap 4B column, and the protein profile of the supernatant before purification was compared with the unbound fraction post-purification via two-dimensional gel electrophoresis (2D-E). The identity of these localized protein spots was confirmed by MS analysis and further analyzed by NanoLC-ESI-QUAD-TOF-MS/MS protein identification. Results indicate that 2 out of 5 protein spots were GSTs with region for class Delta and Epsilon subfamilies similar to other insects; however, the remaining 3 spots did not show any match in the current NCBInr. Both Delta and Epsilon class GSTs are specifically involved in insecticide resistance and their relatively high abundance in the 2D-E map suggests that these enzymes could play a role in the resistance of Meligethes aeneus to the most commonly used pyrethroids. The approach applied in this study for the specific localization of GSTs in 2D-E can be used for similar analyses in other organisms.