Abstract
Wheat is the most important food crop in the world, the unique physiochemical properties of wheat gluten enabling a diverse range of food products to be manufactured. However, genetic and environmental factors affect the technological properties of gluten in unpredictable ways. Although newer proteomic methods have the potential to offer much greater levels of information, it is the older gel-based methods that remain most commonly used to identify compositional differences responsible for the variation in gluten functionality, in part due to the nature of their primary sequences. A combination of platforms were investigated for comprehensive gluten profiling: a QTOF with a data independent schema, which incorporated ion mobility (DIA-IM-MS) and a data dependent acquisition (DDA) workflow using a linear ion trap quadrupole (LTQ) instrument. In conjunction with a manually curated gluten sequence database a total of 2736 gluten peptides were identified with only 157 peptides identified by both platforms. These data showed 127 and 63 gluten protein accessions to be inferred with a minimum of one and three unique peptides respectively. Of the 63 rigorously identified proteins, 26 were gliadin species (4 ω-, 14 α-, and 8 γ-gliadins) and 37 glutenins (including 29 LMW glutenin and 8 HMW glutenins). Of the HMW glutenins, three were 1Dx type and five were 1Bx type illustrating the challenge of unambiguous identification of highly polymorphic proteins without cultivar specific gene sequences. The capacity of the platforms to sequence longer peptides was crucial to achieving the number of identifications, the combination of QTOF-LTQ technology being more important than extraction method to obtain a comprehensive profile. Widespread glutamine deamidation, a post-translational modification, was observed adding complexity to an already highly polymorphic mixture of proteins, with numerous insertions, deletions and substitutions. The data shown is the most comprehensive and detailed proteomic profile of gluten to date.
Highlights
Wheat is arguably the most important grain in the world and forms a staple part of the modern diet (Shewry and Tatham, 2016), being present in many processed foods including breads, noodles, pasta, biscuits, cakes and sauces (Kamal et al, 2009; Gao et al, 2016)
Acetonitrile and water used in chromatography were all HPLC grade (Sigma-Aldrich, Dorset, UK). α-Chymotrypsin (Merck Chemicals, Nottingham, UK) with an activity of ≥300 U/mg and a specific activity 400 U/mg of protein was used for digestion of the gluten proteins
Secondary anti-mouse IgG labeled with alkaline phosphatase and nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro3’indolyphosphate p-toluidine salt (BCIP) substrate solution were sourced from ThermoScientific (Leicestershire, UK)
Summary
Wheat is arguably the most important grain in the world and forms a staple part of the modern diet (Shewry and Tatham, 2016), being present in many processed foods including breads, noodles, pasta, biscuits, cakes and sauces (Kamal et al, 2009; Gao et al, 2016). Its versatility as a food ingredient results from the unique physicochemical properties of the gluten fraction of wheat seed protein. Gluten comprises the major storage proteins of wheat grain, which are traditionally divided into two groups based on their solubility called gliadins and glutenins (Osborne, 1907). The glutenins comprise two groups of subunits, called high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (Bietz and Wall, 1973, 1980), which form alcohol-insoluble polymers stabilized by inter-chain disulfide bonds. When the disulfide bonds are reduced, the glutenin subunits become soluble in aqueous alcohol and amino acid sequences show that gliadins and glutenin subunits are related. It is usual to define both as prolamins, the name originally applied only to wheat gliadins and related proteins from other species
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