Abstract
The wheat gliadins are a complex group of flour proteins that can trigger celiac disease and serious food allergies. As a result, mutation breeding and biotechnology approaches are being used to develop new wheat lines with reduced immunogenic potential. Key to these efforts is the development of rapid, high-throughput methods that can be used as a first step in selecting lines with altered gliadin contents. In this paper, we optimized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC) methods for the separation of gliadins from Triticum aestivum cv. Chinese Spring (CS). We evaluated the quality of the resulting profiles using the complete set of gliadin gene sequences recently obtained from this cultivar as well as a set of aneuploid lines in CS. The gliadins were resolved into 13 peaks by MALDI-TOF-MS. α- or γ-gliadins that contain abundant celiac disease epitopes and are likely targets for efforts to reduce the immunogenicity of flour were found in several peaks. However, other peaks contained multiple α- and γ-gliadins, including one peak with as many as 12 different gliadins. In comparison, separation of proteins by RP-HPLC yielded 28 gliadin peaks, including 13 peaks containing α-gliadins and eight peaks containing γ-gliadins. While the separation of α- and γ-gliadins gliadins achieved by RP-HPLC was better than that achieved by MALDI-TOF-MS, it was not possible to link peaks with individual protein sequences. Both MALDI-TOF-MS and RP-HPLC provided adequate separation of ω-gliadins. While MALDI-TOF-MS is faster and could prove useful in studies that target specific gliadins, RP-HPLC is an effective method that can be applied more broadly to detect changes in gliadin composition.
Highlights
Wheat (Triticum aestivum L.) is a major staple cereal grain consumed by humans worldwide and a major source of protein in the diet
Two ω-5 gliadin peaks in the mass range of about 50–57 kDa were not detected in the gliadin fractions extracted with either 60 or 70% EtOH, but were present in those extracted with 0.15 M NaCl plus EtOH (Figure 1A)
MALDI-TOF-MS separates proteins on the basis of molecular mass and is attractive because the complete analysis of gliadin proteins in a sample can be completed in about 1 min
Summary
Wheat (Triticum aestivum L.) is a major staple cereal grain consumed by humans worldwide and a major source of protein in the diet. The gluten proteins comprise about 70% of the total grain protein (Shewry and Halford, 2002). Some gluten proteins trigger severe diseases in humans, including celiac disease (CD), and food allergies (Biagi et al, 1999; Matsuo et al, 2004, 2005; Battais et al, 2005a; Bittner et al, 2008; Sollid et al, 2012). A number of epitopes that trigger the food allergy wheatdependent exercise-induced anaphylaxis (WDEIA) have been identified in ω-5 gliadins (Battais et al, 2005b), while α-, γ-, and ω-1,2 gliadins contain clusters of epitopes that are active in celiac disease (Tye-Din et al, 2010; Sollid et al, 2012)
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