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The Application of Modern Proteomic and Metabolomic Methodologies to the Assessment of FOOd SAFety

Final Report Summary - FOOSAF (The Application of Modern Proteomic and Metabolomic Methodologies to the Assessment of FOOd SAFety)

The production and consumption of food is central to any society, having important economic, social and in many cases environmental implications. International comparisons have shown that the EU is the world’s largest producer of food and drink products. It is not surprising therefore that the Agro-food sector has major importance/influence on the economy of Europe. The annual production by this industrial sector is 820 billion Euros, constituting over 20% of the total European manufacturing outputs, which makes this sector the leading industry in the EU. Collectively the Agro-food sector supports over 10 million full-time employees, many of which are in small to medium enterprises (SME’s) that contribute to rural development. Thus the economic importance and central role of food in health and the quality of life means that food safety must be of prime interest to the EU and its public authorities, producers and consumers. The activities of this project will impact directly on these multiple sectors of EU industry and society, advancing our means of evaluating and delivering food safety.
The FOODSAFE project illustrates the potential of integration different “omic” datasets and performing mathematical based analysis to improve the characterisation of novel foods. In fact, since the onset of the numerous EU and national programmes used to introduce “omic” technologies into the risk assessments for novel food, major advances in modern molecular breeding have been made. For example, it is now routine to transfer entire pathways into plants generate far greater levels of transgenic products and regulated whole physiological processes. Intragenic methods which use the host plants DNA solely (cisgenic) have been developed and Marker Assisted Selection (MAS) enables the incorporation of multiple traits from sexually compatible species into elite varieties.
Enhancing the levels of nutritional and high value compounds in plants is an important challenge for plant biotechnology. In the present project, a transgenic variety in which fruit antioxidants such as carotenoids, phenolics, tocopherols and ascorbic acid have been elevated simultaneously has been used. The modification has been achieved through a cisgenetic approach, resulting in the down-regulation of the DETIOLATED-1 (DET-1) gene product in a fruit specific manner using the TFM7 promoter. The endogenous gene product is involved in light signalling transduction, and results in the plant believing it is receiving a greater quantity of incident light. In non-European countries such as China and India the commercialisation of GM food crops is underway. This is likely to increase the risk of inadvertent introduction of unauthorised novel foods into the European food chain. In addition a number of industrial products are being development that are derived from GM materials but are enrichment or purified. These products must still go through similar risk assessments as stated by EFSA. In order to introduce and exploit these new varieties or sources into the food chain, safety assessment must be performed. Therefore it is essential that robust, accurate techniques utilising the latest advances are in place to evaluate these novel foods either GM or non-GM. In this sense, EU legislation utilises the concept of substantial equivalence when initially assessing the safety of GM products and/or other novel foodstuffs (EC directive 258/97). Substantiation of equivalence between a new (novel) product and its traditional counterparts can be achieved through the comparison of chemical composition.
The use of ‘omics’ technologies enable a system level overview of the changes arising in the plant as a result of the perturbations. Quantitative proteomics represents an important challenge, as it is the link between the transcriptome and metabolome, where post-translational regulation can arise. In the present project, the biological and biotechnological applicability of a novel label-free quantitative methodology has been demonstrated for the assessment of GM tomato fruit. The analysis of a five-protein mixture at different concentrations to test sample preparation, LC-MS/MS analysis and software tracking performance has demonstrated the linearity, reproducibility, and repeatability of the approach, which has been kept to minimal experimental steps, as shown in the diagram.

Diagrammatic representation of the label-free quantitative proteomic workflow used and its application to the comparative analysis of GM tomato fruit with their wild type (WT) comparator.

Multivariate and pairwise statistical analyses demonstrate that the protein composition of the fruit has been changed through GM, or more precisely altered as a result of manipulating the DET1 target gene product. These perturbations in a representative complement of the cells’ proteomes have occurred, presumably due to the down-stream effects of modulating this regulator of cellular processes associated with light perception. Main differences have been observed in proteins associated with stress response, Calvin-Benson cycle and structural proteins, as well as those participating in glycolysis, lipid metabolism and transferase activity. Thus, from the perspective of food safety and the evaluation of substantial equivalence of novel foods, the cisgenic nutrient dense tomato fruit analysed (TFM7) are not substantially equivalent to their direct comparator. Despite significant variance in the protein composition, however, the individual protein exhibiting the greatest differences between the cisgenic and wild type fruit are known proteins present in plant based foods that have no toxic or allergenic implications. The proteomic analysis could be applied to quantitative judgements relating to the degree of diversity that exists in the market place.
In conclusion, a simple and reliable procedure was optimised for the identification and relative quantitation of tomato fruit proteins from different genotypes. The method uses a workflow based on gel-LC with quantitation relative to internal peptide standards generated from known non-endogenous proteins added to the preparation prior to separation. The utility of the procedure was demonstrated through the analysis of a cisgenic tomato variety engineered to deliver nutrient dense fruit. A comparison with existing transcript data generated on this variety showed good qualitative correlation between transcripts and protein levels but quantitative disparity. The results obtained in terms of linearity, repeatability and reproducibility show that the developed label-free quantitation methodology is ready for validation, subsequent use in food analysis and the integration into multi-level “omic” based characterisation of biological processes.
The outputs from FOODSAFE programme are going to impact directly on a number of key strategic areas including food safety, improved sustainable bio-production and processing, enhanced quality of life, important socio-economic factors, the knowledge based economy, increased competitiveness and prosperity, international development as well as advancing fundamental science and the exploitation of knowledge. In fact, the development of this programme has supposed a scientific advancement as important underpinning fundamental knowledge has been acquired to support the involved activities. This exploitation of knowledge benefits the scientific community and contributes to the progression of science and the multidisciplinary approach of the project ensures that the outputs will appeal to a wide scientific audience. On the other hand, it is well known that through the development of platforms to evaluate our foodstuffs in a more comprehensive manner, food safety, health and food production are improved.
The scientific and technological advances that has supposed the development of this programme potentially impact on European competitiveness creating new and increased markets in the evaluation of foodstuffs leading to increased economic growth and job creation and has a direct route to exploitation providing the opportunity to increase European prosperity. In fact, due to many of the food processors throughout Europe are SMEs located in rural areas, improved food safety evaluation has the potential to develop new industry and opportunity in these areas. This fact will benefit both the Food and Drink industries and Agriculture sector in the development of new varieties and products. The project contributes to the EU’s concept of a knowledge based Bio-Economy, providing the necessary recommendations for the assessment of novel food/feedstuff throughout the EU.

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