Quantification of Gly m 4 Protein, A Major Soybean Allergen, By Two-Dimensional Liquid Chromatography with Ultraviolet and Mass Spectrometry Detection

Abstract

Soybean (Glycine max) is considered a major allergenic food. Gly m 4 is one of several soybean allergens that has been identified to cause an allergic reaction, typically the symptoms are localized effects including the skin, gastrointestinal tract, or respiratory tract. Soybean allergens are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting a range of severity from mild rashes up to anaphylaxis. In this study, we have isolated, purified, and characterized an endogenous Gly m 4 protein. The endogenous protein has 88.0% sequence homology with the theoretically predicted Gly m 4 sequence. Following detailed characterization, an assay was developed for quantification of endogenous Gly m 4 using two-dimensional liquid chromatography with ultraviolet and mass spectrometric detection (2DLC-UV/MS). A linear relationship (R(2) > 0.99) was observed over the concentration range of 12.5-531.7 μg/mL. Over the linear range, the assay recoveries (percent relative error, % RE) ranged from -1.5 to 10.8%. The assay precision (percent coefficient of variation, % CV) was measured at three different Gly m 4 levels on each of the 4 days and did not exceed 11.2%. The developed method was successfully applied to quantify Gly m 4 level in 10 commercial soybean lines. To the best of our knowledge, this represents the first quantitative assay for an intact endogenous Gly m 4 protein.

Assessment of endogenous allergenicity of genetically modified plants exemplified by soybean – Where do we stand?

“…As an example, for the major seed 297 storage protein Gly m 5, consisting of α-, α'and β-subunits, only the α-subunit was measured. 298 Julka et al (2012) studied the natural variability of the Bet v 1 homologue allergen Gly m 4 in 10 299 soybean varieties using 2D liquid chromatography followed by ultraviolet (UV) and MS detection of 300 the intact protein. Gly m 4 quantification in soybean seeds was based on the UV response factor 301 calculated from the purified reference standard of Gly m4, whereas the MS detection confirmed the 302 absence of any co-elution with the Gly m 4 peak during the separation of protein extracts.…”

Using metal nanoparticles as a visual sensor for the discrimination of proteins

“…[1][2][3][4][5][6] Many methods are available for protein detection, such as enzyme-linked immunosorbent assay (ELISA), 7-9 electrophoresis, 10-12 chromatography, [13][14][15] and mass spectrometry (MS). [1][2][3][4][5][6] Many methods are available for protein detection, such as enzyme-linked immunosorbent assay (ELISA), 7-9 electrophoresis, 10-12 chromatography, [13][14][15] and mass spectrometry (MS).…”

Outcome of the public consultation on the draft guidance on allergenicity assessment of genetically modified plants

“…More specifically, an increasing number of publications (that are also not referenced in this draft GD, such as Julka et al, 2012) have shown that there is considerable natural variability in endogenous allergens as a consequence of conventional breeding practices and environmental conditions.…”

“…• By comparison, publications show that endogenous allergen levels are naturally highly variable (up to 19-fold) in conventional varieties. More specifically, an increasing number of publications (that are also not referenced in this draft GD, such as Julka et al, 2012) have shown that there is considerable natural variability in endogenous allergens as a consequence of conventional breeding practices and environmental conditions. • Lack of threshold data on individual soybean allergens for either the sensitization or elicitation phase of an allergic reaction.…”