Comprehensive Proteomic Profiling of Wheat Gluten Using a Combination of Data-Independent and Data-Dependent Acquisition

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.

Wheat Seed Proteins: Factors Influencing Their Content, Composition, and Technological Properties, and Strategies to Reduce Adverse Reactions

“…This number is consistent with the proteomic study of Bromilow et al. () who identified 63 gluten proteins in a single cultivar, using mass spectroscopy and a curated sequence database (Bromilow et al., ). These comprised four ω‐gliadins, 14 α‐gliadins, eight γ‐gliadins, 29 LMW subunits, and eight HMW subunits.…”

“…Allowing for the effects of post-translational modification, we would suggest that the number of gluten proteins present in significant amounts is between 50 and 100. This number is consistent with the proteomic study of Bromilow et al (2017b) who identified 63 gluten proteins in a single cultivar, using mass spectroscopy and a curated sequence database (Bromilow et al, 2017a). These comprised four ω-gliadins, 14 α-gliadins, eight γ -gliadins, 29 LMW subunits, and eight HMW subunits.…”

A curated gluten protein sequence database to support development of proteomics methods for determination of gluten in gluten-free foods

“…Extracted proteins were reduced, alkylated and digested using chymotrypsin and subject to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis as described [42]. LC-MS/MS analysis was carried out using a SYNAPT G2-Si QTOF (quadrupole time of flight) (Waters, Wilmslow, UK) with incorporated ion mobility.…”

Development of reduced gluten wheat enabled by determination of the genetic basis of thelys3alow hordein barley mutant

“…Bands that fall within the region of 30-55 kDa, which make up approximately 70-80% of the total wheat seed storage proteins, are expected to contain α and γ gliadins as well as LMW glutenins, and this was confirmed by the putative identities of the proteins we identified by LC-MS in gel bands excised from this region of the gel (Table S5). Two prominent bands at about 55 kDa, which were drastically reduced in the triple null line were identified as beta-amylase and a LMW glutenin protein [61]. In general, the gliadins and LMW glutenins that are reduced in accumulation in wheat by the triple null WPBF mutations are homologous to the B and C hordeins whose accumulation is reduced by the lys3a mutation in barley.…”