Quantitative detection of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in soybean using two-dimensional gel electrophoresis

Abstract

The Round-up Readyⓡ soybean, which has tolerance against glyphosate, an herbicide, is widely used for soybean processing. Usually, polymerase chain reaction (PCR) is employed to detect GMOs at DNA level. Immunological methods including western-blotting and enzyme linked immunosorbent assay (ELISA) are also used to detect GMOs at protein level. Recently, detection of genetically modified organisms (GMOs) has become more important because of mandatory labeling. The objective of this research was the detection and quantification of CP4 5- enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in GM soybean using two-dimensional gel electrophoresis (2D-PAGE). Purified CP4 EPSPS from cloned E. coli was used as a control to monitor the change of CP4 EPSPS in GM soybean and soybean curd. CP4 EPSPS was detected in GM soybean and GM soybean curd by 2D-PAGE and identified by the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) through databases. CP4 EPSPS in soybean products was resolved on 2D-PAGE according to its characteristic isoelectric point of 5.1 by isoelectric focusing (IEF) and molecular mass of 47.5 kDa by SDS-PAGE. Various percentages CP4 EPSPS in soybean were quantified. The quantitation tests were perfomed on artificial gels with spots varying Gaussian volumes by 2D software program.