Quantitation of Soybean Allergens Using Tandem Mass Spectrometry

Abstract

Soybean (Glycine max) seed contain some proteins that are allergenic to humans and animals. However, the concentration of these allergens and their expression variability among germplasms is presently unknown. To address this problem, 10 allergens were quantified from 20 nongenetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches. Relative quantitation was performed by spectral counting and absolute quantitation was performed using multiple reaction monitoring (MRM) with synthetic, isotope-labeled peptides as internal standards. During relative quantitation analysis, 10 target allergens were identified, and five of these allergens showed expression levels higher than technical variation observed for bovine serum albumin (BSA) internal standard (∼11%), suggesting expression differences among the varieties. To confirm this observation, absolute quantitation of these allergens from each variety was performed using MRM. Eight of the 10 allergens were quantified for their concentration in seed and ranged from approximately 0.5 to 5.7 μg/mg of soy protein. MRM analysis reduced technical variance of BSA internal standards to approximately 7%, and confirmed differential expression for four allergens across the 20 varieties. This is the first quantitative assessment of all major soybean allergens. The results show the total quantity of allergens measured among the 20 soy varieties was mostly similar.

Chemoproteomic Analysis of Intertissue and Interspecies Isoform Diversity of AMP-activated Protein Kinase (AMPK)

“…Relative abundance of the subunit isoforms was assessed by comparing the "spectral counts" of the isoform-unique peptides (Table 1) (37)(38)(39)(40)(41)(42)(43)(44)(45)(46). For instance, in the sample designated healthy hepatocyte-1, ␤1 and ␤2 isoforms were both identified with spectral counts of 6 and 92 (1:15), respectively.…”

“…Likewise, ␤2 was the major isoform in human skeletal muscle but a minor isoform in human heart. Spectral counting has widely been utilized for the relative quantification of proteins in multiple biological samples (37)(38)(39)(40)(41)(42)(43)(44)(45)(46), but its application to the relative quantification of isoforms remains to be validated. In theory, the respective peak intensities of highly homologous peptides should be an ideal measure of the relative abundance of subunit isoforms provided that these peptides are co-eluted and identified in the same gel slice.…”

Detection of Allergen Markers in Food: Analytical Methods

“…Houston et al [216] evaluated the natural variation of ten soy allergens among twenty commercial soybean varieties. Relative quantitation was performed with a spectral counting method referencing bovine serum albumin as an internal standard, and absolute quantitation was performed using an MRM method with isotopically labeled peptide standards.…”

Soybean Allergens: Presence, Detection and Methods for Mitigation

“…Ten allergens were quantified from 20 non-genetically modified commercial soybean varieties using parallel, label-free mass spectrometry approaches (Houston et al, 2011). Also, a multimethod was developed for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction modes.…”