Identification And Quantification Study Of Major Allergenic Proteins In Milk,Peanut,Egg And Soybean By Liquid Phase Tandem High Resolution Mass Spectrometry

Food allergy is an adverse reaction of the body to the ingestion of certain foods.With approximately 220 million people worldwide suffering from food allergies,especially children’s allergies are more prominent,food allergy has become a public health problem with serious health implications.Currently,relying on correct commodity allergen labelling to avoid allergenic foods is the most effective way to protect allergy sufferers and therefore correct commodity allergen labelling is crucial.In order to protect allergic consumers and to verify the accuracy of food allergen labelling,there is an urgent need for a high-throughput,highly sensitive method that can be used to detect different food allergens.In view of this,this paper developed a liquid-phase tandem mass spectrometry（LC-MS/MS）method that can simultaneously identify and quantify the characteristic enzymolysis peptides in multiple allergenic foods,and identified the accuracy of allergen labels of commercial products,with a view to providing data support and reference for the subsequent development of allergen adulteration identification methods.The study consisted of two main parts as follows:In the first part,established a method for the identification and characterisation of the major allergenic proteins in milk,peanut,egg and soybean was based on liquid-phase tandem high-resolution mass spectrometry（LC-Q Orbitrap）combined with proteomics.After extraction,alkylation and enzymatic digestion of the allergenic proteins in the four foods,the formed peptides were sampled and analysed by LC-Q Orbitrap;the collected data were identified and matched with the protein identification software PEAKS Studio8.5 and the online databases NCBI and Uniprot.The results showed that 30,35,25 and 52 proteins were identified in milk,peanut,egg and soybean,respectively;the main allergenic proteins were Bos d 4,Bos d 5,Bos d 6,Bos d 9,Bos d 10,Bos d 11 and Bos d13 in milk;Ara h 1,Ara h 2,Ara h 3 and Ara h 6 in peanut;Gal d 1,Gal d 2,Gal d 3,Gal d4,Gal d 5,Gal d 6 and Gal d 10 in egg;and Gly m 5,Gly m 6 and Gly m 7 in soybean.In addition,the relative abundance of the main proteins in the four food groups was counted and the abundance levels of the allergenic proteins in the total protein were clarified in this study.The test results showed that the major allergens Bos d 9,Ara h 1,Gal d 2 and Gly m6 were suitable for analysis in milk,peanut,egg and soybean,respectively,and for subsequent quantitative analysis by liquid-phase tandem mass spectrometry.In the second part,quantitative studies of the major allergens in milk,peanut,egg and soybean were carried out based on the LC-Q Orbitrap.The peptides were examined in terms of their specificity,response values on mass spectrometry and stability,and their characteristic peptides HQGLPQEVLNENLLR（Bos d 9）,GTGNLELVAVR（Ara h 1）,GGLEPINFQTAADQAR（Gal d 2）and VLIVPQNFVVAAR（Gly m 6）and other auxiliary qualitative peptides were used for quantitative analysis by liquid-phase tandem mass spectrometry.Samples were degreased,protein extracted,alkylated,enzymatically digested and peptide cleaned up before being fed by liquid-phase tandem high-resolution mass spectrometry using parallel reaction detection（PRM）.The ion source parameters,the maximum ion injection time and the collision energy were also systematically optimised for mass spectrometry data acquisition.A linear relationship between the response intensity of the characteristic peptides and the allergen content was established and quantified by external standard method to measure the allergenic protein concentration.In addition,a systematic methodological validation of the established method was carried out to assess the sensitivity,accuracy,precision and stability of the method.The results showed that the established method had an LOD of 1 mg/kg and LOQ of 2.5 mg/kg for milk and egg allergens in biscuit matrix,and an LOD of 2.5 mg/kg and LOQ of 10 mg/kg for peanut and soybean allergens.the recoveries ranged from 82.83% to 105.58%,the intra-day coefficients of variation ranged from 2.6% to 7.5% and the inter-day coefficients of variation The recoveries ranged from 82.83% to 105.58%,the Intraday coefficients of variation ranged from 2.6% to 7.5% and the interday coefficients of variation ranged from3.4% to 7.1%.In addition,the method was successfully applied to the analysis of four allergens in 12 commercially available biscuits,and four products were found to be missing allergen labeling,supporting the urgency of correct allergen labelling..