Mass spectrometry combined with affinity probes for the identification of CP4 EPSPS in genetically modified soybeans.

Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low-abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5-enolpyruvylshikimate-3-phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography-multiple reaction monitoring tandem MS (nano LC-MS/MS-MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)-immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine-containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A-sepharose was used for the affinity capture of ß-conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable-isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time-consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.

Quantitative analysis of genetically modified soya using multiple reaction monitoring mass spectrometry with endogenous peptides as internal standards.

A simple label-free method was developed for the quantification of the herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase using multiple reaction monitoring liquid chromatography-mass spectrometry. Sample pretreatment procedures including ion exchange chromatography and CaCl2 precipitation were used to purify the 5-enolpyruvylshikimate-3-phosphate synthase protein. Quantification of various percentages of genetically modified soya (0.5-100%) was performed by selecting suitable endogenous soybean peptides as internal standards. Results indicated that Gly P (QGDVFVVPR) and Lec P (LQLNK) are useful internal standards for the quantification of low and high percentages of genetically modified soya, respectively. Linear regression analysis of both calibration curves yielded good linearity with R2 of 0.99. This approach is a convenient and accurate quantification method for genetically modified soya at a level as low as 0.5% (less than the current EU threshold for labeling genetically modified soya).

Studying the effect of microwave heating on the digestion process and identification of proteins.

The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.