Research On Soybean Major Allergen Protein Detection Method By LC/MS

Soybean(Glycine Max [L.] Merr.)is rich in protein,oil and other nutrients,and is an important food raw material.It is also rich in allergens,one of the "eight food allergens" recognized by the world health organization(WHO).Glycinin and β-conglycinin are the highest allergen proteins in soybean.At present,there is no effective quantitative method for some allergens,and the relative contents of allergens and their expression variability among soybean varieties are not clear.The main detection methods for soybean allergens are ELISA and PCR,but these two methods have some limitations,such as poor specificity,low detection flux and poor accuracy.In recent years,high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)technology has been widely used in the identification and quantitative analysis of various allergen proteins and their polypeptides in food.This study aims to establish a rapid,sensitive and accurate quasi-deterministic and accurate quantitative detection method for glycinin and β-conglycinin using HPLC-MS/MS technology.The main research contents and results are as follows:(1)A HPLC-MS/MS qualitative determination for eight allergen subunits of glycinin(including G1,G2,G3,G4 and G5 subunits)and β-conglycinin(including α′,α,and β subunits)MS qualitative detection method.By optimizing the extraction conditions,the optimal extraction scheme for soybean protein was finally determined(the buffer was ultra-pure water with a pH of 10.0,the solid-liquid ratio was 1 g∶10 mL,the extraction temperature was 50℃,and the extraction time was 60 min).Soybean holoproteinolytic peptides were analyzed by high resolution mass spectrometry,and peptide section retrieval was completed by using Skyline software and UniProtKB soybean protein database.Two to three specific peptides(total of 21)were screened for 8 allergenic subunits of glycinin and β-conglycinin,of which 10 were reported for the first time in this study.Targeted proteomics tests were performed on 15 negative controls and 24 positive controls in the multi-response monitoring(MRM)mode,and specific validation of the selected signature peptides was completed.Finally,a HPLC-MS/MS method for simultaneous detection of 8 allergenic subunits in soybean was established.(2)A HPLC-MS/MS quantitative detection method for 8 allergen subunits of glycinin and β-conglycinin was established.By optimizing enzymatic hydrolysis conditions,the optimal enzymatic hydrolysis time was determined to be 6 h.The collision energy(CE)and declustering potential(DP)of the signature peptides were optimized to improve the response strength of the MS.The separation efficiency was improved by optimizing the gradient elution conditions of mobile phase in liquid chromatography.The quantitative characteristic peptides of each allergen subunit of glycinin and β-conglycinin were determined,the stable isotopic labeled quantitative specific peptides were designed and synthesized,and the sensitive and accurate HPLC-MS/MS quantitative analysis method of glycinin and β-conglycinin were established.The detection limit(LOD)and quantitative limit(LOQ)of the method were 0.4~200.0 fmol/L and 0.8~400.0 fmol/L,respectively.The recovery rate was 98.75% ~ 110.23%,the in-day coefficient of variation was 2.1% ~ 4.8%,and the diurnal coefficient of variation was 2.6% ~ 3.8%,which could meet the actual testing requirements.(3)The contents of glycinin and β-conglycinin in 128 soybean germplasm were quantitatively analyzed.The whole protein was extracted by the method established in this paper,and the total protein content of the samples was between 283.38 and 420.68 mg/g.The contents of glycinin and β-conglycinin in soybean samples were determined.In the 128 soybean samples,no subunit deletion germplasm was found,and the content of 8 allergen subunits on average were G1 > G2 > α’ > α > β > G4 > G5 > G3.The content of glycinin and β-conglycinin were calculated by summing the contents of each subunit within the range of 102.79~156.36 mg/g and 66.08~121.18 mg/g.The differences among varieties are extremely significant,and the coefficient of variation of the ratio also differs in different ecological intervals.Glycinin and β-conglycinin are both the main allergens of soybean and the key influencing factors of soybean processing characteristics.The quantitative analysis method established in this paper provides a technical basis for soybean allergy threshold research and desensitization treatment,provides technical support for the correct identification of food allergens,and will play a role in the cultivation of low allergenic soybeans and high protein soybean varieties.