A rapid immobilized trypsin digestion combined with liquid chromatography – Tandem mass spectrometry for the detection of milk allergens in baked food

Abstract Cow's milk allergy is the most common food allergy, and caseins are the major allergens in milk. A quantitative proteomics method based on liquid chromatography coupled to mass spectrometry has been widely used in allergen analysis. However, the long period of digestion has limited its use in routine analysis. PHMN-Trypsin (trypsin immobilized on hairy polymer-chain hybrid magnetic nanoparticles), a new type of immobilized trypsin, was used to shorten the digestion time and enhance the digestion efficiency in this study. We developed a rapid digestion method using PHMN-Trypsin to detect milk allergens in baked food by ultrahigh-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS). The PHMN-Trypsin digestion was completed within 15 min, with higher or equal sequence coverage compared to 12–16 h of conventional free trypsin digestion. αs1-casein, αs2-casein, β-casein, and κ-casein were monitored as the allergen proteins. For each target allergen, two or three specific and signature peptides were selected for the UPLC-MS/MS strategy in view of the species specificity, mass response intensity as well as the stability. The limits of quantification of the four proteins in baked foods were 0.38–0.83 μg/g, and their recoveries ranged from 65.2% to 86.1%, with relative standard deviations of less than 11.0%. The method was applied to investigate allergens in commercial baked foods, and the results indicate that this method would be helpful in food allergen screening.