or modification is highly dependent on the peptide sequence, with certain combinations of amino acids being more prone to internal or external chemistry. For all of these reasons, using multiple peptides is likely to provide more accurate results. However, with all of the cleavage and chemistry rules considered, most allergens that we wanted to measure have only a single peptide suitable for quantification.
It has been shown that 50–70% of total seed protein is contributed by the storage proteins glycinin and β-conglycinin with their individual percentages varying drastically between studies (Wolf et al., 1961; Saio, 1969; Murphy and Resurreccion, 1984). In our study, cumulative allergen content was lower than expected, with totals approaching 25% (w/w). We believe this is caused by several factors, including fractional quantitation of all paralogous protein forms and alternative protease cleavage sites.
Quantitation of the β-conglycinin fraction was by far the most incomplete which is largely because peptides for β-conglycinin subunits α′ and β were not analyzed in this study, thereby vastly under-representing the β-conglycinin fraction. Secondly, the peptide used for β-conglycinin α may be under represented due to the presence of an “illegitimate” cleavage site (Keil, 1992) that may in fact be partially cleaved in vitro (Rodriguez et al., 2007). Because this peptide is quantifiable, it is obviously not completely cleaved, and therefore may still be useful for quantification if the AQUA™ peptides are spiked prior to digestion, assuming trypsin is capable of stoichiometrically cleaving this small synthetic peptide. Although this observation would affect the absolute levels of this protein, it should not influence relative levels as the protein would likely be equally processed in vitro. The glycinin fraction was the most prevalent in this study, approaching an additive total of nearly 200 μg/mg (∼20%, w/w). This value is still lower than values provided in the literature (ca. 40%), however a large portion of this discrepancy is likely due to the fact that not all glycinin forms were quantified including glycinin subunit 5 (Gly G5). A previous report on soybean allergens using the same quantitation technology reported substantially lower levels of these storage proteins (Houston et al., 2010). However, the values for that study were for peptide concentrations not proteins which required correction to intact protein molecular mass as listed in the methods. Alternative methods, such as protein ELISA, were not commercially available for the individual allergens in this study.
The levels of allergens have been previously reported to vary between non-GM varieties. For example, in a study of non-GM soybean varieties involving skin reactivity and in vitro IgE binding of 10 soybean cultivars, Codina et al. (2003) reported up to sixfold differences in IgE binding potencies, while Sten et al. (2004) reported wide variation in IgE binding to different varieties of the same species of non-GM crops. To investigate an allergen’s natural variation in expression, Xu et al. (2007) evaluated 16 soybean varieties and showed relative variation in allergens, such as Gly m 5 and Gly m Bd 30K, between wild and cultivated non-GM soybean varieties. Houston et al. (2010) quantified soybean allergens in 20 non-GM varieties and observed anywhere from statistically insignificant changes in expression for some allergens to as much as 10-fold for glycinin G3 (a Gly m 6 isoform) when comparing two different varieties.
These examples give an indication of variation in the allergen levels of various non-GM soybean brought about by breeding. Further research is needed to establish a “baseline” of allergen levels from a composite of both the genetic and even broader environmental factors. Toward this goal, we report here a comparison of genetic versus environmental variation in the expression of eight soybean allergens. While the genetic variation in this study was limited (i.e., early, edible ancestors of soybean were not studied), the environmental compon