Table 1.
Selected wheat marker peptides.
Amino acid sequences of the 16 peptides (P1-16), their specificity for wheat gluten protein types, and the detected peptide scores in the flour.
Table 2.
Optimized LC-MS/MS parameters for the 16 wheat marker peptides.
Multiple reaction monitoring (MRM) parameters of P1-16 and the isotopically labelled peptide standard (*P11) as well as the corresponding response factors (RF), each referred to *P11.
Fig 1.
Schematic diagram showing the development of a method for the quantitation of gluten contents based on peptide yields.
(A) Peptide identification and selection of 16 wheat marker peptides, (B) development of the liquid chromatography tandem mass spectrometry (LC-MS/MS) method with an isotopically labelled peptide as internal standard and optimization of the LC-MS/MS conditions, (C) quantitation of peptide yields in reference gluten protein types and conversion of peptide into protein type and gluten concentrations.
Fig 2.
Precursor to product ion transition (m/z) of each marker peptide (P1-16) and the isotopically labelled standard (*P11).
Marker peptides were quantitated in the respective protein type of wheat (multiple reaction monitoring mode, MRM). Two MRM transitions were monitored for each peptide and the most abundant MRM transition shown here was used for quantitation. HMW-GS, high-molecular-weight glutenin subunits; LMW-GS, low-molecular-weight glutenin subunits.
Table 3.
Limits of detection (LOD) and quantitation (LOQ) for the marker peptides P1-16 in potato flour [μg/g].
Correlation coefficients (r) were determined between peptide concentrations and gluten concentrations in the potato flour spiked to different gluten contents with the wheat flour mixture.
Table 4.
Concentrations of the marker peptides (P1-16) in the respective protein type [μg/g] and the wheat flour mixture [μg/g].
The concentrations of protein types in flour by LC-MS/MS [%] were calculated based on peptide concentrations in the specific protein types and compared to the contents [%] quantitated by RP-HPLC. The contents determined by RP-HPLC were taken as 100% to evaluate the recovery of LC-MS/MS. Protein type concentrations had to be multiplied by the individual correction factor to adjust to recoveries of 100%.
Fig 3.
Linear Pearson correlations between gluten contents and concentrations of peptides from all wheat gluten protein types.
(A) Peptide P4 from low-molecular-weight glutenin subunits (LMW-GS), (B) P7 from high-molecular-weight glutenin subunits (HMW-GS), (C) P8 from γ-gliadins, (D) P11 from α-gliadins, (E) P14 from ω5-gliadins, (F) P15 from ω1,2-gliadins. The presented peptides showed the highest correlation coefficients within the respective protein type (see Table 3).
Table 5.
Concentrations of the marker peptides [μg/g] in seven wheat starches.
The wheat starches used were W4, W6, W8, W11, W13, W14 and W15 as described in Scherf et al [11]. Those marker peptides not listed had concentrations below the respective limit of detection.
Table 6.
Gluten contents [μg/g] of wheat starches W4, W6, W8, W11, W13, W14 and W15.
Results from different methods, LC-MS/MS, GP-HPLC-FLD and R5 ELISA, were compared.