Proteomics in ‘Free-from’ Foods

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Abstract

Wheat is the most agronomically important crop with an annual production of approximately 680 million tonnes per year over the five year period of 2008-2012 (Shewry and Tatham 2016). Wheat typically contributes about 20% of the total calorie intake in Western Europe and between 50-70% in some countries in North Africa and in West and Central Asia. It is estimated that in order to meet the continuous growing global demand wheat production needs to increase by 50% by 2050. Wheat is most commonly consumed as bread, pasta and noodles however it is also used as a food ingredient in other types of foods such as sauces and condiments. The versatility of wheat is largely determined by the unique physiochemical properties of gluten (Bailey 1941). Gluten is one of the earliest proteins to be studied, and was first described by Beccari in 1728 (Bailey 1941) and is readily isolated from wheat flour as a viscoelastic mass. Gluten is a complex mixture of proteins which are the major seed storage proteins found in the cereal grains wheat, barley, rye and oats. Gluten accounts for 70-80% of the total protein content in wheat grains and is traditionally divided into two groups based on their solubility called gliadins and glutenins (Osborne 1907). In genetically pre-disposed patients gluten is able to elicit a non-IgE mediated T-cell response known as coeliac disease (CD). CD affects approximately 1% of the global population for which there is no cure. As no cure is available patients must adhere to a strict gluten-free diet which is often costly and socially excluding. The Codex Standard states that gluten-free foods must contain less than 20 ppm of gluten from wheat, barley, rye and oats and their crossbreeds (FAO/WHO 1983). The Codex Standard also recommends using immunobased methods (or alternative methods) that are able to achieve appropriate sensitivity and specificity for the detection and quantification of gluten with a 10 ppm limit of detection (FAO/WHO 1983). Consequently the current gold standard method for detection of gluten is enzyme linked immunosorbent assay (ELISA) utilising the R5 antibody, however this method is not without shortcomings. Proteomics by mass spectrometry has the potential to offer an alternative, complementary method to determine gluten proteins in foods but for the methodology to become fully validated and accepted it must also overcome similar challenges to immunoassay methods, such as effective extraction of samples and the identification of peptide targets with the requisite specificity. In this research a global approach is taken to aid the development of gluten detection methods using mass spectrometry. One of the major hurdles that has stunted the development of mass spectrometry methods for the detection and quantification for gluten is the lack of protein sequence databases which are required to undertake the MS data searching. In the first results chapter of the thesis a curated gluten protein sequence database was developed (GluPro), and investigated for its utility as a MS data searching tool. It was observed that utilising the GluPro database resulted in improved protein identifications. Following the development of the curated database, extensive method development was carried out to undertake the most extensive background characterisation of the gluten proteome to date using discovery proteomics. To ensure the most comprehensive profile was obtained a number of extraction protocols were investigated and two mass spectrometry platforms with intrinsic differences utilising different modes of acquisition were used. This resulted in the most comprehensive profile of the gluten proteome to date being obtained. In order to meet the continually growing global demand for wheat previous mentioned it is considered that this may be done through the use of genetically modified crops with improved traits such as pesticide resistance. Resulting in the very real possibility of GM crops being introduced into the food supply chain, however there is much widespread public concern regarding the toxicity and allergenicity of genetically modified crops. As wheat is already listed as one of the major eight allergens, it is crucial to be able to undertake safety assessments which are able to assess the toxicity and allergenicity and determine if the GM crop is substantially equivalent to the non-GM counterpart. In the third part of this thesis it is shown how the MS method developed in the previous chapters could be applied and has great potential to be used for safety assessment. Further to this, it is demonstrated how utilising the additional information gathered during the curation of the GluPro database was able to ground the results into in silico measure of toxicity. In the final part of this research all information gathered was interrogated to pick appropriate MRM target peptides, which were unique to a single gluten protein and reproducibly observed to be free from modification. The peptides were synthesised with heavy labels to develop a targeted method to replace current ELISA methods for the detection and quantification. Unexpectedly mass shifts were observed for the precursor ion corresponding to deamidation of the synthetic peptides. Further investigation was undertaken to understand the location and cause of the deamidation sites. This development leads into further recommendation for future development of MRM methods for the detection and quantification of gluten.

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Pen-drive containing supplementary uncurated mass spectrometry data files submitted in pocket inside back cover of print version of thesis

Keyword(s)

gluten; mass spectrometry; proteomics

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