Verification of the cDNA Deduced Sequence of Glutenin Subunit 1Dx5 and an Mr58 000 Repetitive Peptide by Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry (MALDI-MS)

doi:10.1006/jcrs.1999.0292

Journal of Cereal Science

Volume 31, Issue 2, March 2000, Pages 173-183

https://doi.org/10.1006/jcrs.1999.0292 Get rights and content

Abstract

MALDI-MS was used to confirm the cDNA derived amino acid sequence of HMW subunit 1Dx5 and an E. coli -expressed repetitive M_r58 000 peptide based on residues 102–643. Analysis of theM_r 58 000 peptide and of tryptic peptides separated by RP-HPLC showed that the experimentally determinedM_r are consistent, with the calculated values, within the experimental error. This indicates a substantial correctness of the gene derived sequence. In the same way, analysis of tryptic peptides from 1Dx5 allowed about 80% of the sequence to be confirmed, covering residues 1–669 with the exception of two short peptides. There was also good agreement with sequence of the M_r58 000 peptide in the region of identity. The results, giving evidence for a substantial correctness of a large part of the sequence, confirm the absence of extensive glycosylation in subunit 1Dx5 and demonstrate the value of MALDI-MS for direct sequence analysis of HMW subunits and similar proteins.

References (27)

P.R. Shewry et al.
The high molecular weight subunits of wheat glutenin
Journal of Cereal Science
(1992)
S.P. Roels et al.
Evidence for the non-glycoprotein nature of high molecular weight glutenin subunits of wheat
Journal of Cereal Science
(1996)
D.R. Hickman et al.
Molecular weights of high molecular weight (HMW) subunits of glutenin determined by mass spectrometry
Journal of Cereal Science
(1995)
F. Buonocore et al.
Expression and functional analysis of M_r58 000 peptides derived from the repetitive domain of HMW subunit 1Dx5
Journal of Cereal Science
(1998)
P.R. Shewry et al.
Purification and N-terminal amino acid sequence analysis of high molecular weight (HMW) gluten polypeptides of wheat
Biochimica et Biophysica Acta
(1984)
M. Byers et al.
A quantitative comparison of the extraction of protein fractions from wheat grain by different solvents and of the polypeptide and amino acid compositions of the alcohol-soluble proteins
Journal of the Science of Food and Agriculture
(1983)
A.S. Tatham et al.
Structural studies of cereal prolamins, including wheat gluten
In: «Advances in Cereal Science and Technology» (Y. Pomeranz, ed.) Vol. 10, AACC, St Paul, MN
(1990)
P.I. Payne et al.
Structural and genetical studies on the high molecular weight subunits of wheat glutenin
Theoretical and Applied Genetics
(1981)
P.I. Payne
Genetics of wheat storage proteins and the effect of allelic variation on breadmaking quality
Annual Review of Plant Physiology
(1987)
W. Seilmeier et al.
Separation and quantitative determination of high-molecular-weight subunits of glutenin from different wheat varieties and genetic variants of the variety Sicco
Zeitschrift für Lebensmitteluntersuchung und -Forschung
(1991)

N.G. Halford et al.

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L) indicates quantitative effects on grain quality

Theoretical and Applied Genetics

(1992)

P. Reddy et al.

Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker

Theoretical and Applied Genetics

(1993)

K.A. Tilley

Detection of O -glycosidically linked mannose within the structure of a highly purified glutenin subunit isolated from Chinese Spring wheat

Cereal Chemistry

(1997)

Cited by (20)

Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach
2020, Journal of Proteomics
Citation Excerpt :
In particular, most of these studies focused on the characterization of wheat proteins by MS-based methods [5–7]. The aim of these studies is not limited to characterization of the sequence of kernel proteins or to proteomics investigation of cultivars [8–13], but also include efforts to better understand the role of these components in the gluten matrix [14–17]. In spite of its central role for human nutrition, wheat may also cause several adverse reactions and disorders, such as immunoglobulin E (IgE) mediated allergies (celiac disease, CD), which requires a lifelong gluten free diet (GFD) [18], and a less well-defined condition classified as non-celiac wheat sensitivity (NCWS) [19–23].
The close relationship between diet and health is generally recognized and the growing wellness and consciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called “Green-Revolution” in the second half of the 20th century focusing on obtaining properties in terms of higher grain yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein composition of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449.
In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more “natural” and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM-like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these cultivars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption.
The primary structure of wheat glutenin subunit 1Dx2 revealed by electrospray ionization mass spectrometry
2014, Journal of Cereal Science
Citation Excerpt :
Finally, DNA sequences do not provide any information on post-translational modifications, such as glycosylation. Therefore, proteomic techniques using mass spectrometry (MS) have been optimized to verify the available cDNA-deduced sequences of HMW-GS (Foti et al., 2000; Zhang et al., 2008). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-MS has proven to be a sensitive technique to determine the relative molecular mass (Mr) of intact HMW-GS (Zhang et al., 2008).
The high molecular weight glutenin subunits (HMW-GS) play a key role in end-use quality of wheat. Their particular primary structure is mostly derived from DNA sequencing, which gives no information on potential post-translational modifications. This paper reveals the primary structure of HMW-GS 1Dx2 by proteomic analysis. For this purpose, HMW-GS were first isolated from wheat flour (cv. Contra). The relative molecular mass (M_r) of subunit 1Dx2 present in the HMW-GS mixture was then very accurately determined with high-performance liquid chromatography–electrospray ionization-mass spectrometry using a quadrupole-time-of-flight mass analyzer (HPLC–ESI-QTOF-MS). The obtained M_r value (87,105) differed from the value derived from its protein sequence in the NCBI database (87,007). The subunit was further purified by preparative reversed-phase HPLC and partially hydrolyzed with chymotrypsin. The resulting 1Dx2 peptides were then analyzed by HPLC–ESI-MS/MS and the MS data were compared to amino acid sequences in protein databases. The discrepancy between the calculated and the measured M_r of 1Dx2 was explained by a missing proline in the 1Dx2 amino acid sequence from the database and not by any post-translational glycosylation.
Liquid chromatography and mass spectrometry in food allergen detection
2011, Journal of Food Protection
Food allergy is an important issue in the field of food safety because of the hazards for affected persons and the hygiene requirements and legal regulations imposed on the food industry. Consumer protection and law enforcement require suitable analytical techniques for the detection of allergens in foods. Immunological methods are currently preferred; however, confirmatory alternatives are needed. The determination of allergenic proteins by liquid chromatography and mass spectrometry has greatly advanced in recent years, and gel-free allergenomics is becoming a routinely used approach for the identification and quantitation of food allergens. The present review provides a brief overview of the principles of proteomic procedures, various chromatographic set ups, and mass spectrometry instrumentation used in allergenomics. A compendium of published liquid chromatography methods, proteomic analyses, typical marker peptides, and quantitative assays for 14 main allergy-causing foods is also included.
Characterization of Wheat Gluten Proteins by HPLC and MALDI TOF Mass Spectrometry
2008, Journal of the American Society for Mass Spectrometry
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.
Characterization of HMW glutenin subunits in common wheat and related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
2008, Journal of Cereal Science
The sample preparation method of high molecular weight glutenin subunits (HMW-GS) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, without a separation step, by high-performance liquid chromatography (HPLC) was established in this study. Three major factors influencing mass spectra—the ratio of components of the solvent, the resolving time, and the sample volume—were optimized using HMW-GS mixtures extracted from Chinese cultivar Jing 411. The results showed that the optimized method for sample preparation was to resolve HMW-GS from 20 mg in an hour with 50 μl solvent of 0.4% TFA, 30.0% ACN and 69.6% H₂O. The stable mass spectra and accurate molecular weights of 16 major HMW glutenin subunits from common wheat and related species were obtained using the optimized MALDI-TOF-MS method. Seven subunits, where each was from 2–5 cultivars, showed very similar molecular weights. The determined molecular weights of 11 subunits were close to those calculated from their coding sequences. In addition, no positive reaction between HMW-GS and GelCode^® Glycoprotein Staining Reagent was observed. These results suggested that HMW-GS lack extensive post-translational modifications (PTMs), but low levels of glycosylation or phosphorylation present in some subunits cannot be ruled out. Because of its ability to obtain a rapid, complete and precise profile of HMW glutenin subunits without purifying procedures, MALDI-TOF-MS is expected to be a powerful technique for structural and functional studies of HMW glutenin subunits as well as other cereal proteins.
Heterologous expression and protein engineering of wheat gluten proteins
2006, Journal of Cereal Science
A range of systems are available for the production of recombinant wheat gluten proteins, from simple and widely used systems based on Escherichia coli to more sophisticated eukaryotic systems in yeasts or cultured insect cells. The characteristics of these systems are summarised and their advantages and disadvantages for application to wheat gluten proteins discussed. We then review the applications of heterologous expression systems to the synthesis and characterisation of wheat gluten proteins, including the production of wild type and mutant proteins for structure–function studies. We also discuss the use of heterologous expression to establish model systems including perfect repeat peptides based on motifs present in gliadins and glutenin subunits and ‘analogue glutenin proteins’ based on C hordein of barley. It is concluded that the pET series of vectors and E. coli are suitable for most applications, providing high-level expression and being rapid and easy to use.

View all citing articles on Scopus

^f1: Corresponding author: S. Foti. Università degli Studi di Catania, Dipartimento di Scienze Chimiche, Viale A. Doria 6, I-95125 Catania, Italy. Tel: +39 95 7385027; Fax: +39 95 580138; e-mail: [email protected]

View full text

Article preview

Journal of Cereal Science

Abstract

References (27)

The high molecular weight subunits of wheat glutenin

Journal of Cereal Science

Evidence for the non-glycoprotein nature of high molecular weight glutenin subunits of wheat

Journal of Cereal Science

Molecular weights of high molecular weight (HMW) subunits of glutenin determined by mass spectrometry

Journal of Cereal Science

Expression and functional analysis of M_r58 000 peptides derived from the repetitive domain of HMW subunit 1Dx5

Journal of Cereal Science

Purification and N-terminal amino acid sequence analysis of high molecular weight (HMW) gluten polypeptides of wheat

Biochimica et Biophysica Acta

A quantitative comparison of the extraction of protein fractions from wheat grain by different solvents and of the polypeptide and amino acid compositions of the alcohol-soluble proteins

Journal of the Science of Food and Agriculture

Structural studies of cereal prolamins, including wheat gluten

In: «Advances in Cereal Science and Technology» (Y. Pomeranz, ed.) Vol. 10, AACC, St Paul, MN

Structural and genetical studies on the high molecular weight subunits of wheat glutenin

Theoretical and Applied Genetics

Genetics of wheat storage proteins and the effect of allelic variation on breadmaking quality

Annual Review of Plant Physiology

Separation and quantitative determination of high-molecular-weight subunits of glutenin from different wheat varieties and genetic variants of the variety Sicco

Zeitschrift für Lebensmitteluntersuchung und -Forschung

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L) indicates quantitative effects on grain quality

Theoretical and Applied Genetics

Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker

Theoretical and Applied Genetics

Detection of O -glycosidically linked mannose within the structure of a highly purified glutenin subunit isolated from Chinese Spring wheat

Cereal Chemistry

Cited by (20)

Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach

The primary structure of wheat glutenin subunit 1Dx2 revealed by electrospray ionization mass spectrometry

Liquid chromatography and mass spectrometry in food allergen detection

Characterization of Wheat Gluten Proteins by HPLC and MALDI TOF Mass Spectrometry

Characterization of HMW glutenin subunits in common wheat and related species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

Heterologous expression and protein engineering of wheat gluten proteins

Regular ArticleVerification of the cDNA Deduced Sequence of Glutenin Subunit 1Dx5 and an Mr58 000 Repetitive Peptide by Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry (MALDI-MS)

Abstract

Journal of Cereal Science

Journal of Cereal Science

Journal of Cereal Science

Journal of Cereal Science

Biochimica et Biophysica Acta

A quantitative comparison of the extraction of protein fractions from wheat grain by different solvents and of the polypeptide and amino acid compositions of the alcohol-soluble proteins

Journal of the Science of Food and Agriculture

Structural studies of cereal prolamins, including wheat gluten

In: «Advances in Cereal Science and Technology» (Y. Pomeranz, ed.) Vol. 10, AACC, St Paul, MN

Structural and genetical studies on the high molecular weight subunits of wheat glutenin

Theoretical and Applied Genetics

Genetics of wheat storage proteins and the effect of allelic variation on breadmaking quality

Annual Review of Plant Physiology

Separation and quantitative determination of high-molecular-weight subunits of glutenin from different wheat varieties and genetic variants of the variety Sicco

Zeitschrift für Lebensmitteluntersuchung und -Forschung

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L) indicates quantitative effects on grain quality

Theoretical and Applied Genetics

Analysis of a genomic DNA segment carrying the wheat high-molecular-weight (HMW) glutenin Bx17 subunit and its use as an RFLP marker

Theoretical and Applied Genetics

Detection of O -glycosidically linked mannose within the structure of a highly purified glutenin subunit isolated from Chinese Spring wheat

Cereal Chemistry

Regular Article
Verification of the cDNA Deduced Sequence of Glutenin Subunit 1Dx5 and an M_r58 000 Repetitive Peptide by Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry (MALDI-MS)