ie University, Australia
[edited-by] Reviewed by: Bronwyn Jane Barkla, Universidad Nacional Autónoma de México, Mexico; Benjamin Schwessinger, University of California Davis, USA; Roque Bru-Martinez, Universidad de Alicante, Spain
Correspondence: *Correspondence: Gregory S. Ladics, DuPont Agricultural Biotechnology, Pioneer Hi-Bred, Wilmington, DE 19880, USA. e-mail: email@example.com
[other] This article was submitted to Frontiers in Plant Proteomics, a specialty of Frontiers in Plant Science.
Soybeans accumulate protein and oil during development, making the mature soybean seed a valuable commodity. For 2008/09, the farm value of soybeans in the United States neared $30 billion (http://www.ers.usda.gov/Briefing/SoybeansOilCrops/), exceeded in value only by corn. The value of soybean meal as both a food and feed source is dependent on the character of its storage protein reserves. In soybean, the most abundant storage proteins are members of the cupin superfamily; the legumins (glycinins), and vicilins (β-conglycinins; Derbyshire et al., 1976; Kinney et al., 2001; Takahashi et al., 2003; Radauer and Breiteneder, 2007), which account for approximately 70% of total seed protein. Together with cotton and maize, soybeans represent the majority of the genetically modified (GM) products developed to date. Most of the soybeans grown in the United States are GM varieties. In 2011, about 94% of all soybeans cultivated for the commercial market in the United States were GM (USDA National Agricultural Statistics Service (NASS), 2011). Soybean is considered one of the “big eight” allergenic foods in the United States. Soybean allergens are grouped into five categories: seed storage proteins Gly m 5 (β-conglycinin) and Gly m 6 (glycinin); Gly m TI (Kunitz trypsin inhibitor), Gly m Bd 28K, and Gly m Bd 30K (P34; FARRP version 10 allergen database, 2010: Ogawa et al., 2000; L’Hocine and Boye, 2007). One of the concerns involving GM soybeans, particularly in the European Union, is the potential increase in the levels of such endogenous allergens compared to those obtained with traditional breeding methods and subsequent enhancement in their sensitization or elicitation capacity. Data, however, are lacking in regard to the natural variability of endogenous allergen levels in non-GM soybean (Doerrer et al., 2010). Variability in protein expression levels may result from genetic differences (Fehr et al., 2003), environmental differences (Murphy and Resurreccion, 1984; Maestri et al., 1998), nutrient stress (Gayler and Sykes, 1985; Paek et al., 1997), use of different breeding methods (Burton, 1989; Yaklich, 2001; Krishnan et al., 2007), or interaction between genotype and the environment (Paek et al., 1997; Piper and Boote, 1999). These data are critical for describing and understanding potential differences of protein allergen levels among GM and non-GM soybean varieties (Doerrer et al., 2010).
Much of what we know about the protein component of mature soybean has come from various offline separation techniques like polyacrylamide gel electrophoresis, with or without isoelectric focusing, coupled with antibody or dye based detection methods. Two-dimensional (2-D) gels utilizing appropriate immobilized pH gradients that zoom to either acidic or basic pH ranges have been very successful at separating acidic and basic subunits of glycinin as well as the α and β subunits of β-conglycinin (Mooney et al., 2004; Hajduch et al., 2005; Natarajan et al., 2006; Danchenko et al., 2009). Achieving separation allows for quantification of the various spots using densitometry methods. Ultimately, these gel methods rely on mass spectrometry to distinguish the highly related proteins present in the various spots, which in the case of seed storage proteins exceed 100 (Agrawal et al., 2008). The multiplicity of 2-D spots that must each be accounted for in a quantitative study coincides with the biggest problem of 2-D gel-based quantification – low throughput (Rabilloud et al., 2010).
Quantification of proteins by mass spectrometry can be achieved using a te