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[Figure ID: F1]
Experimental design and processing workflows. (A) Each cultivar (92B12, 92M10, 92M72, and 93B15) was grown in each location (NE, IA, PA, ONT, KS, and GA) in triplicate using a randomized complete block design for the plots. Different states/provinces in the map are colored to distinguish the three major climate zones represented within the chosen growth regions. Stars within each state/province indicate the location where the soybean cultivation occurred. (B) Three hundred grams of soybean was harvested from each block (each replicate). The replicate material was pooled and ground, with 10 seeds worth of meal portioned for analysis. Protein was extracted in quadruplicate from each, and analyzed using multiple reaction monitoring with AQUA™ peptide internal standards. Absolute quantitative values for soybean allergens and anti-nutritional factors were calculated and compared for variance between cultivars and environments (locations).
[Figure ID: F2]
Environmental and varietal variation. Percent coefficients of variation (%CV, standard deviation(−mean × 100) for allergen quantities are plotted for each allergen with respect to environment and cultivar. The variation was normalized with respect to the mean. This variation was also analyzed using ANOVA, presented in Table 1.
[Figure ID: F3]
Absolute quantities for allergens and anti-nutritional proteins. The absolute quantities for the eight proteins of interest are presented. Data are grouped by cultivar with each separate bar representing the quantity of the protein for the indicated environment (location). Error bars are standard deviation between extraction replicates.
[Figure ID: F4]
Allergen totals by location. Values presented are the sum totals for all eight proteins measured. Data are grouped by location, and error bars are summed standard deviations for each allergen. Groups were ordered according to their average allergen sums.
[Figure ID: F5]
Environment versus cultivar biplot. Results of principal component analysis were visualized using a biplot. The x axis represents the direction of maximum variation through the data, which is called the first principle component direction. The y axis represents the direction of the next highest variation through the data, which is orthogonal to the first principle component and called the second principle component direction. Text in the body of the plot represents the samples. The name for each has two parts: location abbreviation followed by cultivar number. Correlations between allergens are represented by vectors terminating with text indicating the allergen they represent. Angles between these vectors, acute or obtuse, represent positive or negative correlation respectively.
[Figure ID: FA1]
Matrix dilution series. The linearity of response of endogenous peptides was assessed with constant AQUA™. A twofold dilution series from 2 to 0.125 μg/injection of soybean (Williams 82) digest was analyzed. The fractional changes, from undiluted through the twofold dilution series, both measured (via response ratio) and actual, were plotted to assess the slope and correlation values. Values on the x and y axes are the fraction of undiluted sample (2 μg/injection) for each successive dilution in the series (1, 0.5, 0.25, and 0.125 μg/injection). Technical variation for the experiment was