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

Wheat gluten proteins are key determinants of baking quality. While untargeted proteomics enables relative quantification, absolute quantities of specific gluten protein groups are not available so far. We developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) stable isotope dilution assay to quantify eleven individual gluten protein groups based on isotope-labeled internal standards corresponding to 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 a multiple advanced generation intercross wheat population comprising 394 inbred lines. 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) 2-4) 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.