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)

Jaime H Mejías; Xiaoqiao Lu; Claudia Osorio; Jeffrey L Ullman; Diter von Wettstein; Sachin Rustgi

doi:10.3390/nu6041578

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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)

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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)

Jaime H Mejías, Xiaoqiao Lu, Claudia Osorio, Jeffrey L Ullman, Diter von Wettstein and Sachin Rustgi

Nutrients, Vol.6(4), pp.1578-1597

04/15/2014

DOI: https://doi.org/10.3390/nu6041578

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https://hdl.handle.net/2376/106575

PMCID: PMC4011052

PMID: 24739977

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Abstract

Celiac Disease - etiology

Celiac Disease - pathology

Chromatography, High Pressure Liquid - methods

Glutens - analysis

Chemical Fractionation

Gliadin - adverse effects

Electrophoresis, Polyacrylamide Gel

Glutens - adverse effects

Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods

Gliadin - analysis

Triticum - chemistry

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.

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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)
Creators: Jaime H Mejías - Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. mejias.j@gmail.com
Xiaoqiao Lu - Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. xiaoqiao.lu@wsu.edu
Claudia Osorio - Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. cosorio72@gmail.com
Jeffrey L Ullman - Department of Agricultural and Biological Engineering, University of Florida, Gainesville, FL 32611, USA. jullman@ufl.edu
Diter von Wettstein - Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. diter@wsu.edu
Sachin Rustgi - Department of Crop & Soil Sciences, Washington State University, Pullman, WA 99164, USA. rustgi@wsu.edu
Publication Details: Nutrients, Vol.6(4), pp.1578-1597
Academic Unit: Crop and Soil Sciences, Department of
Publisher: Switzerland
Grant note: R01 GM080749 / NIGMS NIH HHS GM080749-01A2 / NIGMS NIH HHS 2R42DK072721-02 / NIDDK NIH HHS R42 DK072721 / NIDDK NIH HHS
Identifiers: 99900547051401842
Language: English
Resource Type: Journal article