[Allergenic protein content in high-molecular-weight glutenin from genetically edited wheat by ultra-performance...

To establish a qualitative and quantitative method for detecting allergenic proteins in wheat based on the high-molecular-weight(HMW) glutenin Tri a 26, and to analyze and compare the allergenic protein content between genetically edited wheat and conventional control wheat samples.

The allergenic protein Tri a 26 was selected as the target protein, and its specific peptide IFWGIPALLK was identified and artificially synthesized. Wheat samples were homogenized and extracted with 100 mmol/L Tris-HCl buffer(pH 8.5) containing 4 mol/L urea and 0.1 mol/L dithiothreitol. After alkylation of cysteine residues, the extracts were digested with trypsin at 37 ℃ for 16 h. Quantification was performed using multiple reaction monitoring in positive-ion mode via dart spray ionization, with an external standard method. The Tri a 26 content in genetically edited wheat and conventional control wheat was measured and compared.

The standard solution of the specific hydrolyzed peptide exhibited good linearity in the range of 10-2000 ng/mL(r>0.999). The limit of quantification(LOQ) for the IFWGIPALLK peptide in the sample was 0.328 ng/g. The spike recovery rates at three concentration levels ranged from 84.8% to 95.4%(n =6), with relative standard deviations(RSDs) between 2.99% and 6.12%. The Tri a 26 content in genetically edited wheat and conventional control wheat ranged from 1.976 to 2.069 mg/g.

This method demonstrates high sensitivity and specificity and can be effectively applied to the detection of HMW glutenin in wheat. The study found that the Tri a 26 content in genetically edited wheat was not higher than that in conventional control wheat.