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Absolute quantification of gluten protein groups and their relation to wheat baking quality

ABSTRACT: Wheat gluten proteins are key determinants of baking quality. While untargeted proteomics enable relative quantification, absolute quantities of specific gluten protein groups are not available so far. We developed a targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS) stable isotope dilution assay (SIDA) to quantify eleven individual gluten protein groups based on isotope-labeled internal standards corresponding to the selected marker peptides. The comparison of targeted and untargeted measurements revealed differences in protein composition, likely arising from different MS/MS acquisition strategies and protein assignment. We correlated the absolute protein content with baking quality traits in the Bavarian multiple advanced generation intercross wheat population. None of the individual groups correlated strongly with any baking quality trait. Six groups (α-gliadin 2, γ-gliadin 1, low-molecular-weight glutenin subunit (LMW-GS) 3, and high-molecular-weight glutenin subunits (HMW-GS) 1–4) showed weak to moderate associations (r = 0.32–0.64), mainly with grain protein content, sedimentation value, and wet gluten content. LMW-GS 3 represents the rare i-type containing eight cysteine residues. Loaf volume was only weakly to moderately correlated, primarily with HMW-GS 1 (r = 0.41) and HMW-GS 3 (r = 0.40), supporting the superior effect of Dy10. By contrast, HMW-GS 5 (Dx2, Dx5) showed little effect, consistent with a stronger influence of y-type glutenin subunits. Summing up the protein content across groups increased correlation strengths, yet baking quality remains a complex trait shaped by multiple proteins and non-protein factors.

ORGANISM(S): Triticum Aestivum

SUBMITTER: Christine Kaemper

PROVIDER: PXD072549 | panorama | Wed Dec 31 00:00:00 GMT 2025

REPOSITORIES: PanoramaPublic

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Project description:The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from six days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21-51% at 37 DPA. Thus, the concentration of some specific peptides wasere reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. , evidenced by a notable absence of the predicted peptides despite near complete protein sequence coverage. In addition to a suite of previously characterised hordeins, two novel barley B-hordein isoforms and two avenin-like proteins (ALPs), mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins are due tocomprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. Hordeins are members of the gluten protein family and consumption can trigger health conditions such as coeliac disease, non-coeliac gluten sensitivity, and gluten allergy. It is therefore important to study the accumulation of these proteinshordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food reduced coeliac reactivity.processing properties.

Project description:Within the complex wheat flour proteome, the gluten proteins have attracted most of the attention because of their importance in determining the functional properties of wheat flour doughs and their roles in human health conditions such as celiac disease and food allergies. However, certain non-gluten proteins also trigger immunological responses but may be present in flour in low amounts or obscured by the more abundant gluten proteins in two-dimensional gels of total protein preparations. Non-gluten proteins were preferentially extracted from the flour with a dilute salt solution and separated by two-dimensional gel electrophoresis. Proteins in 172 gel spots were identified by tandem mass spectrometry after cleavage with trypsin or chymotrypsin. Fifty-seven different types of non-gluten proteins were identified, including 14 types that are known or suspected immunogenic proteins. The predominant proteins in 18 gel spots were gluten proteins. Transgenic wheat lines in which specific groups of gluten proteins were suppressed by RNA interference were used to estimate the amount of carry-over of gluten proteins in the salt-soluble protein fraction. Analysis of salt-soluble proteins from a transgenic line missing omega-1,2 gliadins demonstrated that certain omega-1,2 gliadins were present in large amounts in the salt-soluble fraction and obscured relatively small amounts of beta-amylase and protein disulfide isomerase. In comparison, analysis of a transgenic line in which alpha gliadins were absent revealed that glyceraldehyde-3 phosphate dehydrogenase was a moderately abundant protein that co-migrated with several alpha gliadins. The proteomic map of the non-gluten protein fraction of wheat flour developed in this study complements a proteomic map of the total flour proteins developed previously for the same cultivar. Knowing the identities of low abundance proteins in the flour as well as proteins that are hidden by some of the major gluten proteins on two-dimensional gels is critical for studies aimed at assessing the immunogenic potential of wheat flour and determining how the growth conditions of the plants affect the levels of specific immunogenic proteins in the flour.

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Absolute quantification of gluten protein groups and their relation to wheat baking quality

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