Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens

• Published in: Food and chemical toxicology Vol. 116; no. Pt B; pp. 207 - 215
• Main Authors: Lu, Mei, Jin, Yuan, Cerny, Ron, Ballmer-Weber, Barbara, Goodman, Richard E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.06.2018
• Subjects: agglutinins; allergenicity; allergens; Allergens - blood; Antigens, Plant - blood; beta-conglycinin; binding proteins; Blotting, Western - methods; Chromatography, Liquid; embryogenesis; Food Hypersensitivity - immunology; gels; genetic engineering; Globulins; Glycine max; Glycine max - immunology; Glycinin; Humans; IgE; immunoglobulin E; Immunoglobulin E - metabolism; Kunitz-type proteinase inhibitor; LC-MS/MS; mass spectrometry; Mass Spectrometry - methods; quantitative analysis; risk assessment; Seed Storage Proteins - blood; soy protein; Soybean allergens; Soybean Proteins - blood; soybeans; spectrometers; Tandem Mass Spectrometry; toxicology; Trypsin Inhibitor, Kunitz Soybean - metabolism; Two-dimensional immunoblots; β-conglycinin; SBP; MOWSE; IgE; Two-dimensional immunoblots; WHO/IUIS; Glycinin; MW; SKTI; CHAPS; β-conglycinin; LC-MS/MS; OECD; vdc; LEP; SPT; NCBI; DBPCFC; GE; Soybean allergens
• ISSN: 0278-6915; 1873-6351; 1873-6351
• DOI: 10.1016/j.fct.2018.04.032

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either β-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety. [Display omitted] •Subunits of β-conglycinin (Gly m 5) and glycinin (Gly m 6) were major soybean allergens.•Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein in four out of ten subjects.•Soy agglutinin, seed biotinylated, late embryogenesis and sucrose-binding proteins bound IgE of soy-sensitized individuals.•Many putative soybean allergens were not detected as IgE binding proteins among the ten subjects.