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Development of sensitive and robust mass spectrometric methods for detection of GM material in food ingredients.

Project Code: G01023

31/08/2005

School of Biological Sciences, Royal Holloway, University of London
Bramley, P

Aims and Objectives

The project's principal aim was to test the feasibility of using mass spectrometry (MS) to identify the GM proteins found in the most common GM crops, i.e. soya, maize and tomato. It was also designed to establish if the technique would be sensitive enough to detect GM food at the levels required by the EU (0.9%). The precise objectives were:

Production and digestion of recombinant GM proteins to obtain accurate masses of GM biomarker peptides.
Optimisation of protein extraction from GM and non-GM soya, maize and tomato.
Protein digestion and peptide separation conditions for GM proteins/peptides in soya, maize and tomato.
Mass spectrometry of GM peptides using MALDI-TOF and ESI MS/MS.
Assessment of the detection sensitivity in GM food products.
Feasibility study for the use of ion trap mass spectrometry as an alterative to MALDI and ESI MS/MS.

Approach

The experimental approach used a combination of traditional biochemical techniques for protein purification hyphenated with state of the art MS to detect and identify the GM proteins in soya, maize and tomato. Once this was achieved, spiked foods were tested for these GM proteins, using MS, and also quantified the amounts of these proteins in the foods quantified. The limits of sensitivity were also established in each case. Three types of MS were compared for their applicability to identify and quantify GM proteins: ion trap MS matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionisation quadrupole time-of-flight (nano ESI-QTOF) MS. Finally, quantification was established using both amine-specific labelling reagents (iTRAQ) and stable isotope labelled peptides (AQUA).

Experimental approach

The GM soya and maize proteomes were fractionated by gel filtration, anion exchange chromatography and SDS-PAGE prior to MS. The protein fractions enriched in EPSPS were combined and subjected to proteolysis using trypsin, endoprotease AspN and Glu-C. The resultant peptides were subjected to each of the three MS techniques and peptides identified by comparison to databases. Once identified, we prepared spiked samples of bread, Pot Noodle and sausages to establish if the EPSPS could be detected in foods as well as in raw materials. In addition, quantification was determined by the use of two technologies; aminespecific labelling reagents (iTRAQ) and stable isotope labelled peptides (AQUA). Transgenic tomatoes, with the CrtI (phytoene desaturase) transgene were analysed for the present of the CRTI protein by MS procedures

Key findings

We have established that MS can detect, identify and quantify GM proteins in GM soya, maize and tomato. It can also be used to detect the GM proteins in food matrices such as sausages and bread. Overall, 75 % and 71% sequence coverage of 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS), the transgenic protein in soya and maize, was obtained using trypsin, endoprotease AspN and Glu-C protein digestions. The identification was also accomplished at <0.9 % GM soybean seeds, which is the current EU threshold for foodlabeling requirements. Preliminary semi purification of the proteomes was necessary in order to obtain fractions enriched in the GM protein in all cases.

Technical evaluation and interpretation

The analytical strategy was based on enzymatic digestion of the GM protein and its identification by mass spectrometry. However, enrichment of the transgenic protein in the extract was essential, since major storage proteins present in crude extracts suppressed its detection. The approach that yielded most efficient purification of CP4 EPSPS employed gel filtration and anion exchange chromatography, followed by SDS-PAGE. CP4 EPSPS peptide identification was obtained by MALDI-TOF MS, nano LC-nano ESI-QTOF MS and MS/MS and interrogation of protein sequence databases. The analytical approach was shown to identify the transgenic protein CP4 EPSPS from 0.9 % GM soybean seeds, which is the current European Union labelling threshold for GM food and crops. This method is scalable and offers the prospect to be adapted to other food matrices or to other transgenic proteins from different GM maize and soya preparations.

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