chnique called multiple reaction monitoring (MRM), during which signals from the endogenous protein are compared to those from a synthetic heavy-labeled internal standard (Kirkpatrick et al., 2005; Brun et al., 2009; Houston et al., 2010; Stevenson et al., 2010). More specifically, MRM analyses monitor peptides from proteins of interest, which are specific products of proteolysis often generated using the enzyme trypsin. The internal standards are synthetic peptides identical to the endogenous peptides of interest that have been labeled by the addition of a heavy isotope-containing amino acid, thereby changing its mass but nothing else. Because MRM is a form of tandem mass spectrometry (MS/MS), peptides (precursors) are fragmented during the analysis. Fragmentation is used to verify the sequence of the specific peptide of interest, which provides an additional level of specificity to the analysis. This technology is aided greatly by genome and RNA sequencing efforts that provide the sequences for proteins of interest, which allows for rapid MRM method development. Using this technology, proteins that differ in sequence by a single non-isobaric amino acid are theoretically discernable. In addition, MRM analysis using nanospray ionization is highly reproducible with replicate analyses, having less than 15% variation on average including analyses at the limits of instrument detection (Addona et al., 2009).
Recently, to begin to understand the natural variation of the allergenic protein levels in soybean, Houston et al. (2010) utilized tandem mass spectrometry to characterize the natural variation of 10 allergens (representing all five categories of soybean allergens identified to date) in 20 commercially available non-GM soybean varieties. The authors reported that the absolute quantities [absolute per unit protein (μg allergen/mg protein)] of the studied allergens extended over a 10-fold range. The objective of the current study is to further evaluate the influence of genotypes and environments. Four non-GM commercially available varieties of soybeans were each grown at six different locations (Georgia, Iowa, Kansas, Nebraska, Ontario, and Pennsylvania, Figure 1). Soybean allergens were measured similarly to Houston et al. (2010) and absolute quantities of soybean allergens reported. Principal Component Analysis (PCA) was conducted to explore the patterns of multivariate data and a biplot (Gabriel, 1971) was utilized to provide results graphically. Genotypic variation and environmental variation were quantified relative to the overall mean and relative to residual variation using Analysis of Variance (ANOVA)-based F-test. The data suggest that over a broad geographical region, the environment plays a larger role than genotype in determining allergen/anti-nutrient protein levels.
Unless stated otherwise, all reagents were purchased through Research Products International, Mount Prospect, IL, USA.
Four commercially available non-GM soybean varieties (92B12, 92M10, 92M72, and 93B15 were utilized for this study. A similarity matrix using all the markers available that are in common between the varieties was conducted and it was found that the lines have anywhere from 62 to 71% similarity. The study design included six sites located in the commercial soybean-growing regions of North America: Quitman, GA, USA; Richland, IA, USA; Larned, KS, USA; York, NE, USA; Branchton, ON, Canada; and Hereford, PA, USA. The field phase of this study was conducted during the 2007 growing season. The mean temperature during the day for each site was as follows: GA (90.5°F); IA (81.5°F); KS (86.5°F); NE (80.3°F); ON (77.5°F); PA (86.8°F). The mean temperature during the night for each site was as follows: GA (68.2°F); IA (60.5°F); KS (59.2°F); NE (58.7°F); ON (53.5°F); PA (58.2°F). Average rainfall for each site is as follows: GA (4.98″); IA (4.79″); KS (2.23″); NE (3.45″); ON (1.99″); PA (2.87″). A randomized complete block design containing three blocks, with each block containing the 92B12, 92M10, 92M72, and 93B15 commercial soybean varieties,