2DLC-UV/MS Assay for the Simultaneous Quantification of Intact Soybean Allergens Gly m 4 and Hydrophobic Protein from Soybean (HPS)

Abstract

Top-down approaches for quantification of proteins based on separation and mass spectrometric assays hold promise due to their high specificity and avoidance of both proteolytic steps and need for generation of monoclonal antibodies. In this study, a 2DLC-UV/MS assay was developed for the simultaneous quantification of two intact soybean allergens, hydrophobic protein from soybean (HPS) and Gly m 4. Both of these allergens were purified from soybean seeds followed by complete characterization. The method validation consisted of evaluating linearity, precision, and recovery. A linear relationship (R(2) > 0.99) between concentrations of the two proteins and their respective peak areas was observed over the concentration ranges from 6.9 to 355.1 μg/mL and from 11.9 to 599.8 μg/mL for Gly m 4 and HPS, respectively. For the 4 day validation study, precision range (%CV) was observed to be from 4.7 to 9.2% for HPS and from 6.3 to 9.4% for Gly m 4. The assay recovery range (%RE) was observed to be from -1.1 to -13.7% for HPS and from -3.5 to 15.2% for Gly m 4. The assay was applied on 10 non-transgenic commercial lines to quantify the relative levels of the two allergens. The HPS and Gly m 4 levels ranged from 64 to 479 μg/g and from 204 to 637 μg/g, respectively. To the best of the authors' knowledge, this represents the first 2DLC-UV/MS assay for the simultaneous quantitation of selected allergens at the intact level.

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

“…Using the same methodology and soybean varieties, in another work by the same group the Gly m 1 308 and Gly m 4 allergens were simultaneously quantified, resulting in 7.5 and 3-fold differences, 309 respectively (Kuppannan et al, 2014). 310…”

Recent update on methodologies for extraction and analysis of soybean seed proteins

“…While gel staining methods are still most commonly used for protein detection, MS‐based labelling and label‐free methods are increasing in popularity. Kuppannan et al . used 2D LC with UV diode array and MS detection for the assay and quantification of intact soybean allergens.…”

“…While gel staining methods are still most commonly used for protein detection, MS-based labelling and label-free methods are increasing in popularity. Kuppannan et al 78 used 2D LC with UV diode array and MS detection for the assay and quantification of intact soybean allergens. The authors reported the linearity, precision and recovery of relative levels of soybean allergen Gly m 4 and hydrophobic protein from ten non-transgenic soybeans.…”

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“…Thus, currently there is zero tolerance. Use of MS-based proteomic techniques for quantification of allergens in food shows an increasing trend and tendency to overcome the problem of expensive equipment and the need for highly qualified personnel; LC-MS-based approaches are the fastest growing analytical methodologies in food analysis [43].…”