Discovery and screening of thermostable signature peptides for robust quantification of wheat allergens in processed foods

Wheat gluten is a primary cause of food-related disorders such as wheat allergy and celiac disease, yet its reliable quantification in processed foods remains challenging. To address this, we developed a data-independent acquisition (DIA) proteomic screening approach to discover heat-resistant marker peptides, identifying QYEQQPVVPSK, LEGSDALSTR, and VNVPLYR as thermostable signature peptides for quantifying high molecular weight glutenin (HMW-G) and low molecular weight glutenin (LMW-G). Their specificity was confirmed by BLAST analysis against other cereal proteomes. Based on these peptides, we developed a UHPLC-MS/MS method, which demonstrated exceptional sensitivity with limits of detection (LOD) of 0.081 mg/kg for HMW-G and 0.049 mg/kg for LMW-G and limits of quantification (LOQ) of 0.4 mg/kg and 0.244 mg/kg, respectively, in potato chips and surimi. This method, representing the first application of thermostable glutenin peptides, has been successfully applied to complex processed products, providing a robust tool for allergen detection. • Reports the first rationally designed thermostable signature peptides for glutenin. • Validates an ultra-sensitive UHPLC-MS/MS method for gluten detection. • Achieves a detection limit far below the 20 mg/kg gluten-free threshold. • Successfully uncovers hidden wheat allergens in complex food matrices.