Abstract: In order to develop an accurate method to detect the major soybean allergens using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the signature peptides of the five main soybean allergen proteins were selected and verified. Total protein was extracted from samples under optimized conditions, and it was hydrolyzed by sequencing-grade trypsin. The resulting hydrolysate was analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) combined with computer search of the Uniprot database. Targeted identification of peptide fragments of the five major allergens (Glycinin subunits G1, G2, G3, G4 and G5, β-conglycinin α’, α and β subunits, Gly m Bd 30K, Gly m Bd 28K, and Kunitz trypsin inhibitors) in 24 soybean cultivars was performed. Finally a total of 29 signature peptides with high response value and good reproducibility were obtained, and they were verified by high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS) in the multiple reaction monitoring (MRM) mode. The results showed that the specificity, sensitivity and stability of these peptides were in line with the requirements of mass spectrometry detection. In conclusion, this study provide a theoretical basis and technical support for the comprehensive and accurate detection of soybean allergens in foods.

Key words: soybean; allergen; signature peptide; ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry; high performance liquid chromatography-triple quadrupole mass spectrometry