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Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.
PLoS One. 2013; 8(3):e58682.Plos

Abstract

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.

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Authors+Show Affiliations

Lagrain B
German Research Center for Food Chemistry, Freising, Germany. bert.lagrain@biw.kuleuven.be
Brunnbauer M
No affiliation info available
Rombouts I
No affiliation info available
Koehler P
No affiliation info available

MeSH

GliadinGlutensMolecular WeightProteomeSpectrometry, Mass, Electrospray IonizationTriticum

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23520527

Citation

Lagrain, Bert, et al. "Identification of Intact High Molecular Weight Glutenin Subunits From the Wheat Proteome Using Combined Liquid Chromatography-electrospray Ionization Mass Spectrometry." PloS One, vol. 8, no. 3, 2013, pp. e58682.
Lagrain B, Brunnbauer M, Rombouts I, et al. Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry. PLoS One. 2013;8(3):e58682.
Lagrain, B., Brunnbauer, M., Rombouts, I., & Koehler, P. (2013). Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry. PloS One, 8(3), e58682. https://doi.org/10.1371/journal.pone.0058682
Lagrain B, et al. Identification of Intact High Molecular Weight Glutenin Subunits From the Wheat Proteome Using Combined Liquid Chromatography-electrospray Ionization Mass Spectrometry. PLoS One. 2013;8(3):e58682. PubMed PMID: 23520527.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry. AU - Lagrain,Bert, AU - Brunnbauer,Markus, AU - Rombouts,Ine, AU - Koehler,Peter, Y1 - 2013/03/08/ PY - 2012/10/12/received PY - 2013/02/05/accepted PY - 2013/3/23/entrez PY - 2013/3/23/pubmed PY - 2013/9/11/medline SP - e58682 EP - e58682 JF - PloS one JO - PLoS One VL - 8 IS - 3 N2 - The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/23520527/Identification_of_intact_high_molecular_weight_glutenin_subunits_from_the_wheat_proteome_using_combined_liquid_chromatography_electrospray_ionization_mass_spectrometry_ L2 - https://dx.plos.org/10.1371/journal.pone.0058682 DB - PRIME DP - Unbound Medicine ER -