Stable Isotope Dilution Mass Spectrometry For Comprehensive Quantification Of Seafood Allergens

As a source of high-quality protein,the consumption of seafood has increased rapidly,which has led to the increasing prevalence of seafood allergy and has become a serious global public health problem.Labeling food allergens on food packaging is the most effective way to help allergic people avoid allergens.However,the existing detection methods for seafood allergens have poor reproducibility and low accuracy.They can only detect one allergen at a time,which is challenging to meet the detection requirements of complex food matrices and simultaneous quantification of multiple allergens in practical applications.This study screened the characteristic peptides of three allergenic proteins(parvalbumin,tropomyosin,and arginine kinase)of two specific sensitizing kinds of seafood(fish and crustaceans)by targeted proteomics.Based on the isotope dilution mass spectrometry technology,the quantitative confirmation technology for the above three allergenic proteins and the detection method that can simultaneously identify and quantify a variety of allergens have been further established and applied to the detection of seafood products allergenic proteins in complex processed foods.The main research contents are as follows:(1)Parvalbumin(PV),a typical sensitizing protein of fish,was taken as the research object in this study.The peptide fingerprints of cod PV were obtained through ultra-high-resolution mass spectrometry analysis.The candidate signature peptides LFLQNFSAGAR and VFEIIDQDK were screened from species specificity,enzyme digestion efficiency,stability,amino acid sequence length,and mass spectrometry characteristics.The quantitative confirmation of PV in a complex matrix was realized by optimizing the liquid chromatography and MS parameters and pretreatment conditions of the distinct peptide segment,combined with isotope dilution mass spectrometry technology.The methodological validation showed that the quantitative confirmation method was accurate and precise.(2)In this study,Tropomyosin(TM)and Arginine Kinase(AK),the specific sensitizing proteins of crustaceans,were used as the research objects.The peptide fingerprints of TM and AK were obtained through ultra-high-resolution mass spectrometry analysis.The peptides ALSNAGEVAALNR,LEDELVNEK,and FLAEEADR were screened for TM as candidate signature peptides.The peptides LTSAVNEIEK and VSSTLSSLEGELK were screened for AK as candidate signature peptides.The simultaneous quantitative confirmation of TM and AK in a complex matrix was realized by optimizing the liquid chromatography and MS parameters of signature peptides and pretreatment conditions and combining them with isotope dilution mass spectrometry technology.The methodological validation showed that the quantitative confirmation method was accurate and precise.In addition,aiming at the problem that the matrix effect affects the accuracy of the technique,studies on the validation of the spiked recovery rate of different types of food matrices have been carried out,and the results show that the matrix effect has little impact on the accuracy of the method.(3)Given the need for simultaneous quantitative confirmation of multiple allergens in practical application scenarios,this study has established a simultaneous qualitative and quantitative detection method for multiple seafood allergens(fish and crustaceans)based on optimizing the liquid quality conditions and pretreatment conditions.The results of methodological validation show that the two allergens present a good linear regression relationship within the concentration range of 0.1-1000 ng/m L.The LOD and LOQ are lower than the requirements of the sensitization threshold.The recovery rate was 78.53%-111.58%in flour,and the intra-day and intra-day variation coefficients were less than 15%,indicating that the accuracy and precision of this method were good.The characteristic of simultaneous quantification of multiple allergens shortens the detection time.Using this method to detect can ensure accuracy and achieve the purpose of saving costs and increasing efficiency.