Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix‐assisted laser desorption/ionisation mass spectrometry and high‐performance liquid chromatography/electrospray ionisation mass spectrometry

Cunsolo, Vincenzo; Foti, Salvatore; Saletti, Rosaria; Gilbert, Simon; Tatham, Arthur S.; Shewry, Peter R.

doi:10.1002/rcm.938

Cited by 47 publications

(29 citation statements)

References 19 publications

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“…The use of MALDI-MS for mapping tryptic peptides of HMW subunits demonstrated good agreement between MS measured molecular weights and those derived from the gene sequences [11,12]. Tryptic digests have also been analyzed using ESI mass spectrometry in MS and MS/MS modes to confirm amino acid sequences of HMW subunits [12]. In this study we demonstrate the viability of detailed characterization and identification of wheat gluten proteins by RP-HPLC followed by high-resolution MALDI mass spectrometry (MS and MS/MS) of the resulting protein fractions and their trypsin peptide digests.…”

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confidence: 74%

“…MALDI-MS of purified HMW subunits yielded molecular mass determinations consistent with their cDNA derived amino acid sequences [10]. The use of MALDI-MS for mapping tryptic peptides of HMW subunits demonstrated good agreement between MS measured molecular weights and those derived from the gene sequences [11,12]. Tryptic digests have also been analyzed using ESI mass spectrometry in MS and MS/MS modes to confirm amino acid sequences of HMW subunits [12].…”

mentioning

confidence: 86%

“…In spite of this, wheat proteins have been widely studied using HPLC It. 8], and mass spectrometry techniques are now widely applied alone or in combination with other chromatographic techniques in proteomic studies of wheat [9][10][11][12]. Previous studies have established that MALDI-TOF MS can resolve a large number of wheat gliadins and glutenin subunits [9].…”

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confidence: 99%

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

Qian

Preston²,

Krokhin

et al. 2008

J. Am. Soc. Mass Spectrom.

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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 w-gliadin fractions (FI-4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5-8),59 in the a-and l3-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9-31) and 5 peaks in the v-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 Dyl0 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type 1, 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. . The complexity of these patterns can be attributed to the presence of two or three sets of homologous chromosomes in durum and common wheat} respectively, and to additional polymorphism related to mutation of gluten protein genes into many allelic forms [3]. Gliadin patterns have been extensively used to provide definitive identification of all but the most closely genetically related varieties [1]. Specific peaks or patterns, particularly those of the HMW glutenin subunits, have been Address reprint requests to Dr. W. Ens, TOF Laboratory, 506 Allen Bldg, Department of Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. E-mail: ens@cc.umanitoba.ca associated with differences in processing performance among wheat classes and varieties, allowing their use as quality markers [4][5][6][7].There are several difficulties associated with proteomic analysis of wheat storage proteins, including the limited databases available, the limited number of basic residues (particularly in the LMW glutenin subunits), the complexity resulting from the presence of sets of homologous proteins} and the presence of repeating motifs. In spite of this, wheat proteins have been widely studied using HPLC It. 8], and mass spectrometry techniques are now widely applied alone or in combination with other chromatographic techniques in proteomic studies of wheat [9][10][11][12]. Previous studies have established that MALDI-TO...

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confidence: 74%

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confidence: 86%

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confidence: 99%

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

Qian

Preston²,

Krokhin

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…BGel-free^systems such as reversed-phase high-performance liquid chromatography (RP-HPLC) and high-performance capillary electrophoresis (HPCE) were also developed and broadly utilized (Gao et al 2010). Various mass spectrometry (MS) methods were used to characterize HMW-GS and LMW-GS (Cunsolo et al 2003;Foti et al 2000;Lagrain et al 2013;Liu et al 2010). MS appears to be as effective as the more conventional methods for which it differs in terms of resolution, sensitivity, accuracy, throughput and quantification which can increase their applicability to the durum wheat food chain.…”

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confidence: 99%

Gel-Based and Gel-Free Analytical Methods for the Detection of HMW-GS and LMW-GS in Wheat Flour

et al. 2015

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Durum wheat (Triticum turgidum L.) flour is instrumental for the production of pasta worldwide. The quality of this food rests on flour processing and on its protein content and composition. Gluten proteins as high and low-molecular weight glutenins (GS) are important to predict the flour technological property in pasta making. Different methods were compared to separate, identify and quantify GS in flours from two wheat cultivars. Sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) gave in a fast way information about the GS assets. Two-dimensional gel electrophoresis (2D-GE) allowed for the highest resolution in detecting and quantifying single GS, subsequently identified by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS). Reversed-phase high-performance liquid chromatography (RP-HPLC) is a non-gel alternative system for separation and quantification of single GS that when combined with matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF/MS) gave information about their exact masses. This method gives also quantitative indications of each individual GS. Different GS patterns and contents were detected in the flour of the two cultivars, underlining the importance of these analytical methods before determining the best flour processing procedure in pasta making. The different methods were evaluated with a modular approach consisting of a grid of different parameters and a non-linear score within each module.

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“…They found the motif QXP was positively favored as a deamidation site. The amino acid structure of HMW-GS-1Dy10 has been determined [13]. Examination of the sequence reveals QLP, QIP and QHP once each, QEP twice and QQP 28 times.…”

Section: Introductionmentioning

confidence: 99%

Antibodies to Wheat High-Molecular-Weight Glutenin Subunits in Patients with Celiac Disease

Ellis

Lozano-Sánchez

Redondo

et al. 2012

Int Arch Allergy Immunol

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Background: Wheat gluten comprises gliadins and glutenins. The high-molecular-weight (HMW) glutenin subunits (GS)-1Dy10 are toxic for patients with celiac disease (CD). This study aimed to assess whether CD patients mount a serological response to HMW-GS-1Dy10. Methods: Recombinant HMW-GS-1Dy10 was deamidated using human recombinant tissue transglutaminase. MALDI-TOF was performed to compare the level of deamidation of glutamine residues between material before and after treatment. Enzyme-linked immunosorbent assays were developed. Sera from patients with untreated CD and gastrointestinal disease controls were tested and receiver operator characteristics were used to calculate cutoffs. Results: MALDI-TOF revealed a number of fragments matching known HMW-GS-1Dy10 sequences within both the deamidated and non-deamidated material. Evidence of deamidation of glutamine residues was found only within the human transglutaminase-treated material. Patients with untreated CD had significantly increased levels of serum antibodies to HMW-GS-1Dy10 compared to controls. Undeamidated HMW-GS-1Dy10 IgA antibodies had sensitivities and specificities of 72.5 and 78.26%, respectively. Deamidated HMW-GS-1Dy10 IgA antibodies had sensitivities and specificities of 76.8 and 65.2%. Undeamidated HMW-GS-1Dy10 IgG antibodies had sensitivities and specificities of 75.3 and 68.1%. Deamidated HMW-GS-1Dy10 IgG antibodies had sensitivities and specificities of 36.2 and 92.8%. Conclusion: Patients with untreated CD have raised antibody levels to HMW-GS-1Dy10, indicating the participation of these proteins in the adaptive immune response to gluten. Discrimination between CD patients and controls is not enhanced by deamidation of HMW-GS-1Dy10. Thus antibodies to these proteins are not useful markers for CD detection.

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Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix‐assisted laser desorption/ionisation mass spectrometry and high‐performance liquid chromatography/electrospray ionisation mass spectrometry

Cited by 47 publications

References 19 publications

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

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

Gel-Based and Gel-Free Analytical Methods for the Detection of HMW-GS and LMW-GS in Wheat Flour

Antibodies to Wheat High-Molecular-Weight Glutenin Subunits in Patients with Celiac Disease

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