Comparison of MALDI-TOF-MS and RP-HPLC as Rapid Screening Methods for Wheat Lines With Altered Gliadin Compositions

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.

Genome-Wide Identification, Characterization and Expression Pattern Analysis of the γ-Gliadin Gene Family in the Durum Wheat (Triticum durum Desf.) Cultivar Svevo

“…The RP-HPLC analysis allowed a good separation of the peaks in the γand ω-gliadin zones, whereas the peaks corresponding to the α/β gliadins were difficult to separate (Figure 6b). This difficulty has already been reported for bread wheat and can be explained by the grand average of the hydropathicity index (GRAVY) that was found to be much more similar among α-gliadins than γand ω-gliadins [33]. Furthermore, in this case, at least six peaks were distinguishable in the chromatographic zone corresponding to the γgliadin fraction (Figure 6b).…”

Biomolecular Profiling by MALDI-TOF Mass Spectrometry in Food and Beverage Analyses

“…The procedure was found to be reliable, fast and robust; the accuracy of classification was 97%, and the results were independent of the storage of flour and extracts as well as a possible exchange of instrument operators [ 88 ]. Recently, MALDI-TOF MS was compared with reversed-phase high performance liquid chromatography (RP-HPLC) for analysis of gliadins from Chinese Spring (CS) wheat, which indicated advantages and limits of the methods [ 89 ]. These proteins were isolated from a standard CS wheat and its aneuploid lines with altered gliadin composition and measured with SA as a matrix using the double layer deposition technique.…”