Characterization of wheat gluten proteins by hplc and MALDI TOF mass spectrometry

  • Yuwei Qian
  • Ken Preston
  • Oleg Krokhin
  • Jean Mellish
  • Werner EnsEmail author


We have performed a detailed characterization and identification of wheat gluten proteins obtained from the Teal variety of Canadian hard red spring wheat. RP-HPLC separation of the sample into 35 fractions has reduced the spectral complexity; this was followed by MALDI mass spectrometry (MS), which showed the presence of six or fewer resolved protein components above 20 kDa in each RP-HPLC fraction, giving a total of 93 MS resolved peaks. These included 17 peaks in the ω-gliadin fractions (F1–4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5–8), 59 in the α- and β-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9–31) and 5 peaks in the γ-gliadin fractions (F32–35). Peptide maps of tryptic digests of HPLC fractions were obtained from a tandem quadrupole time-of-flight mass spectrometer (MALDI QqTOF MS) and were submitted to the ProFound search engine. HMW glutenin subunits including Ax2*, Dx5, Bx7, and Dy10 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type I, were identified with reasonable sequence coverage from HPLC fraction 5, 7, 17, and 18. The identities of the peptides attributed to selected gluten proteins were confirmed using MS/MS with BioMultiView to match the predicted and measured partial amino acid sequences. Because of incomplete wheat DNA databases, many wheat gluten proteins could not be identified. These results suggest that the combination of RP-HPLC with MS and MS/MS techniques is a promising approach for the characterization of wheat gluten proteins.


Gluten Protein Glutenin Subunit High Molecular Weight Glutenin Subunit HPLC Fraction Bread Wheat Variety 
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  1. 1.
    MacRitchie, F. Physiocochemical Properties of Wheat Proteins in Relation to Functionality. Adv. Food Nutr. Res. 1992, 36, 1–87.CrossRefGoogle Scholar
  2. 2.
    Bietz, J. A.; Simpson, D. G. Electrophoresis and Chromatography of Wheat Proteins: Available Methods and Procedures for Statistical Evaluation of the Data. J. Chromatogr. 1992, 624, 53–80.CrossRefGoogle Scholar
  3. 3.
    Payne, P. I. Genetics of Wheat Storage Proteins and the Effect of Allelic Variation on Bread-Making Quality. Ann. Rev. Plant Physiol. 1987, 38, 141–153.CrossRefGoogle Scholar
  4. 4.
    Cornish, G. B.; Skylas, D. J.; Siriamornpun, S.; Békés, F.; Larroque, O. R.; Wrigley, C. W.; Wootton, M. Grain Proteins as Markers of Genetic Traits in Wheat. Aust. J. Agric. Res. 2001, 52, 1161–1171.CrossRefGoogle Scholar
  5. 5.
    Dworschak, R. G.; Ens, W.; Standing, K. G.; Preston, K. R.; Marchylo, B. A.; Nightingale, M. J.; Stevenson, S. G.; Hatcher, D. Analysis of Wheat Gluten Proteins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. J. Mass Spectrom. 1998, 33, 429–435.CrossRefGoogle Scholar
  6. 6.
    Mann, M.; Hendrickson, R. C.; Pandey, A. Analysis of Proteins and Proteomes by Mass Spectrometry. Annu. Rev. Biochem. 2001, 70, 437–473.CrossRefGoogle Scholar
  7. 7.
    Shewry, P. R.; Tatham, A. S. The Prolamin Storage Proteins of Cereal Seeds: Structure and Evolution. Biochem. J. 1990, 267, 1–12.Google Scholar
  8. 8.
    Marchylo, B. A.; Hatcher, D. W.; Kruger, J. E.; Kirkland, J. J. Reversed-Phase High-Performance Liquid Chromatographic Analysis of Wheat Proteins Using a New, Highly Stable Column. Cereal Chem. 1992, 69, 371–378.Google Scholar
  9. 9.
    Godovac-Zimmermann, J.; Brown, L. R. Perspectives for Mass Spectrometry and Functional Proteomics. Mass Spectrom. Rev. 2001, 20, 1–57.CrossRefGoogle Scholar
  10. 10.
    Hickman, D. R.; Roepstorff, P.; Shewry, P. R.; Tatham, A. S. Molecular Weights of High Molecular Weight Subunits of Glutenin Determined by Mass Spectrometry. J. Cereal Sci. 1995, 22, 99–103.CrossRefGoogle Scholar
  11. 11.
    Foti, S.; Maccarrone, G.; Saletti, R.; Roepstorff, P.; Gilbert, S.; Tatham, A. S.; Shewry, P. R. Verification of the cDNA Deduced Sequence of Glutenin Subunit 1Dx5 and an M-r 58,000 Repetitive Peptide by Matrix-Assisted Laser Desorption/Ionization Mass spectrometry (MALDI-MS). J. Cereal Sci. 2000, 31, 173–183.CrossRefGoogle Scholar
  12. 12.
    Cunsolo, V.; Foti, S.; Saletti, R.; Gilbert, S.; Tatham, A.; Shewry, P. R. Structural Studies of Glutenin Subunits 1Dy10 and 1Dy12 by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and High-Performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 442–454.CrossRefGoogle Scholar
  13. 13.
    Loboda, A. V.; Krutchinsky, A. N.; Bromirski, M.; Ens, W.; Standing, K. G. A. Tandem Quadrupole/Time-of-Flight Mass Spectrometer with a Matrix-Assisted Laser Desorption/Ionization Source: Design and Performance. Rapid Commun. Mass Spectrom. 2000, 14, 1047–1057.CrossRefGoogle Scholar
  14. 14.
    Dachkevitch, T.; Redaelli, R.; Biancardi, A. M.; Metakovsky, E. V.; Pogna, N. E. Genetics of Gliadins Coded by the Group 1 Chromosomes in the High-Quality Bread Wheat Cultivar Neepawa. Theor. Appl. Genet. 1993, 86(2/3), 389–399.Google Scholar
  15. 15.
    Werner, W. E. Ferguson Plot Analysis of High Molecular Weight Glutenin Subunits by Capillary Electrophoresis. Cereal Chem. 1995, 72, 248–251.Google Scholar
  16. 16.
    Seilmeier, W.; Valdez, I.; Mendex, E.; Wieser, H. Comparative Investigations of Gluten Proteins from Different Wheat Species: II. Characterization of Omega-Gliadins. Eur. Food Res. Technol. 2001, 212, 355–363.CrossRefGoogle Scholar
  17. 17.
    Zhang, W.; Chait, B. ProFound: An Expert System for Protein Identification Using Mass Spectrometric Peptide Mapping Information. Anal Chem. 2000, 72, 2482–2489.CrossRefGoogle Scholar
  18. 18.
    Halford, N. G.; Field, J. M.; Blair, H.; Urwin, P.; Moore, K.; Robert, L.; Thompson, R.; Flavell, R. B.; Tatham, A. S.; Shewry, P. R. Analysis of HMW Glutenin Subunits Encoded by Chromosome 1A of Bread Wheat (Triticum aestivum L.) Indicates Quantitative Effects on Grain Quality. Theor. Appl. Genet. 1992, 83, 373–378.CrossRefGoogle Scholar
  19. 19.
    Shewry, P. R.; Halford, N. G.; Tatham, A. S. The High Molecular Weight Subunits of Wheat Glutenin. J. Cereal Sci. 1992, 15, 105–120.CrossRefGoogle Scholar
  20. 20.
    Ikeda, T. M.; Nagamine, T.; Fukuoka, H.; Yano, H. Characterization of New Low Molecular-Weight Glutenin Subunit Genes in Wheat. Theor. Appl. Genet. 2002, 104, 680–687.CrossRefGoogle Scholar
  21. 21.
    Masci, S.; D’Ovidio, R.; Lafiandra, D.; Kasarda, D. D. Characterization of a Low-Molecular-Weight Glutenin Subunit Gene from Bread Wheat and the Corresponding Protein that Represents a Major Subunit of the Glutenin Polymer. Plant Physiol. 1998, 118, 1147–1158.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Yuwei Qian
    • 1
  • Ken Preston
    • 2
  • Oleg Krokhin
    • 1
  • Jean Mellish
    • 2
  • Werner Ens
    • 1
    Email author
  1. 1.TOF Laboratory, 506 Allen Bldg, Department of Physics and AstronomyUniversity of ManitobaWinnipegCanada
  2. 2.Grain Research LaboratoryCanadian Grain CommissionWinnipegCanada

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