title: Analysis of wheat prolamins, the causative agents of celiac sprue, using reversed phase high performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).
abstract: Wheat prolamins, commonly known as "gluten", are a complex mixture of 71-78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%-60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed.
authors.affiliation: Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. mejias.j@gmail.com.
authors.forename: Jaime H
authors.initials: JH
authors.lastname: Mejías
authors.affiliation: Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. xiaoqiao.lu@wsu.edu.
authors.forename: Xiaoqiao
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. cosorio72@gmail.com.
authors.forename: Claudia
authors.initials: C
authors.lastname: Osorio
authors.affiliation: Department of Agricultural and Biological Engineering, University of Florida, Gainesville, FL 32611, USA. jullman@ufl.edu.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Ullman
authors.affiliation: Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. diter@wsu.edu.
authors.forename: Diter
authors.initials: D
authors.lastname: von Wettstein
authors.affiliation: Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. rustgi@wsu.edu.
authors.forename: Sachin
authors.initials: S
authors.lastname: Rustgi
mesh_terms: D002446:Celiac Disease / Q000209:etiology / Q000473:pathology; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005903:Gliadin / Q000009:adverse effects / Q000032:analysis*; D005983:Glutens / Q000009:adverse effects / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014908:Triticum / Q000737:chemistry*
chemical_list: D005983:Glutens; D005903:Gliadin; C037302:glutenin
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