 was utilized at each site. Each soybean variety was planted in a two-row plot, which was bordered on either side by one row of non-GM commercial soybean variety of similar relative maturity. Sites were surrounded by at least 10 feet of bare ground buffer area. All plants grew well at all locations and harvesting was conducted at typical maturity/dryness levels. Typical would refer to 95% of pods on the plant are mature and seed weight adjusted to 13% seed water content. Seed samples for each cultivar (300 g from each block) were harvested and pooled. There was no morphology/physiology differences among the seeds collected from the different locations. The pools were ground and a portion removed for each cultivar. Samples were shipped at ambient temperatures to the University of Missouri for analysis. Samples were stored at room temperature in the dark until extracted.
Protein was isolated in quadruplicate from 0.25 g of soybean meal using the extraction method described in (Stevenson et al., 2009). Briefly, soybean meal was suspended in 2.5 mL of Tris-buffered phenol (pH 8.0; Fisher Scientific, Inc., Pittsburgh, PA, USA) and 2.5 mL extraction buffer [0.9 M sucrose, 0.1 M Tris-HCl pH 8.0, 10 mM EDTA, 0.4% (v/v) 2-mercaptoethanol]. The mixture was homogenized using a T25 Basic S1 Disperser (IKA Works, Inc., Wilmington, NC, USA) on speed 4 with the S25N-10 tool. The emulsion was inverted at 4°C for 2 h and then centrifuged at 5000 × g and 4°C for 20 min. Phenol was removed and a single back extraction was performed in the same manner. Phenol fractions were combined and protein precipitated using five volumes of 95% (v/v) methanol with 0.1 M ammonium acetate and incubation at −20°C overnight. Precipitated protein was pelleted by centrifugation at 5000 × g and 4°C for 5 min, and washed by re-suspending in 10 mL fresh methanol-ammonium acetate solution three times, 80% (v/v) acetone two times, and 70% (v/v) ethanol once, with 20 min incubations at −20°C before pelleting. Protein was stored in 80% (v/v) acetone at −20°C.
Twenty-five microliters of precipitated protein was collected by centrifugation at 16,000 × g at 4°C for 1 min. Pellets were dissolved in 300 μL of urea buffer (50 mM Tris-HCl, pH 8.0, and 8 M urea) by pipetting and vortexing, and finally centrifuged at 16,000 × g at 4°C for 10 min to pool and pellet insoluble material. Supernatants were collected, split into two equal portions and snap frozen in liquid nitrogen and stored at −80°C. Dissolved protein samples were thawed at 4°C, and vortexed to make homogenous. Samples were quantified in quadruplicate using 3 μL of undiluted sample per replicate. Quantification was accomplished using the Coomassie dye-binding assay at 1× (Bio-Rad, Hercules, CA, USA) using the 96-well plate format employing an in-house-prepared soy protein (Williams 82) standard at 2 mg/mL in urea buffer. A five point standard curve of 0 (3 μL of urea buffer), 1, 2, 4, and 8 mg/well was created using a twofold dilution series made with urea buffer. The standard curve used 4 μL/well, and was done in triplicate. Final protein concentration was the average of the protein concentrations obtained from the four replicate assays. After the concentrations were determined, the reconstitution step was repeated using back-calculated values for volume (using between 200 and 800 μL/sample) in order to reconstitute each sample to a concentration of 1–2 mg/mL. Protein quantification was repeated in the same manner, using one of the frozen aliquots, to verify the final concentrations were between 1 and 2 mg/mL.
The remaining aliquot of frozen protein was thawed at 4°C, vortexed to make homogenous and 10 μg of each sample was portioned into 1.5 mL polypropylene tubes. For each, the volume was brought to 10.0 μL using 8 M urea buffer. Disulfide bonds were reduced with 15.0 μL of reduction solution (16.7 mM dithiothreitol (DTT), 6.7 ng/μL BSA in 50 mM ammonium bicarbonate) for 1 h at room temperature. Reduced cysteines were carboxyamidomethylated with 5.0 μL alkylation solution (300 mM iodoacetamide in 50 mM ammon