Proteomics of gluten: mapping of the 1Bx7 glutenin subunit in Chinese Spring cultivar by matrix‐assisted laser desorption/ionization

Alberghina, Gaetano; Cozzolino, Rosaria; Fisichella, Salvatore; Garozzo, Domenico; Savarino, Andrea

doi:10.1002/rcm.2032

Cited by 22 publications

(13 citation statements)

References 27 publications

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“…The deduced molec- ular masses were 85,782 Da (1Dx5 *t ) and 87,663 Da (1Dx5.1 *t ), which were consistent with those determined by MALDI-TOF-MS, and the differences were within the limits of experimental error in the mass range of HMW glutenin (Hickman et al 1995). This suggested that both subunits lacked extensive post-translational modifications, such as glycosylation and phosphorylation, as did other HMW-GSs analyzed by different proteome approaches (Cozzolino et al 2001;Cunsolo et al 2004;Alberghina et al 2005). The two novel subunit genes were deposited in the GenBank under accession nos.…”

Section: Identification Of Novel Hmw-gs In Ae Tauschii: Sds-page Anasupporting

confidence: 74%

Novel x-Type High-Molecular-Weight Glutenin Genes From Aegilops tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

Zhang

Wang

et al. 2008

Genetics

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Two new x-type high-molecular-weight glutenin subunits with similar size to 1Dx5, designated 1Dx5 *t and 1Dx5.1 *t in Aegilops tauschii, were identified by SDS-PAGE, RP-HPLC, and MALDI-TOF-MS. The coding sequences were isolated by AS-PCR and the complete ORFs were obtained. Allele 1Dx5 *t consists of 2481 bp encoding a mature protein of 827 residues with deduced M r of 85,782 Da whereas 1Dx5.1 *t comprises 2526 bp encoding 842 residues with M r of 87,663 Da. The deduced M r 's of both genes were consistent with those determined by MALDI-TOF-MS. Molecular structure analysis showed that the repeat motifs of 1Dx5 *t were correspondingly closer to the consensus compared to 1Dx5.1 *t and 1Dx5 subunits. A total of 11 SNPs (3 in 1Dx5 *t and 8 in 1Dx5.1 *t ) and two indels in 1Dx5 *t were identified, among which 8 SNPs were due to C-Tor A-G transitions (an average of 73%). Expression of the cloned ORFs and N-terminal sequencing confirmed the authenticities of the two genes. Interestingly, several hybrid clones of 1Dx5 *t expressed a slightly smaller protein relative to the authentic subunit present in seed proteins; this was confirmed to result from a deletion of 180 bp through illegitimate recombination as well as an in-frame stop codon. Network analysis demonstrated that 1Dx5 *t , 1Dx2 t , 1Dx1.6 t , and 1Dx2.2 * represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in an associated star-like phylogeny, suggesting that there were at least four independent origins of hexaploid wheat. In addition to unequal homologous recombination, duplication and deletion of large fragments occurring in Glu-D-1-1 alleles were attributed to illegitimate recombination.

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Section: Identification Of Novel Hmw-gs In Ae Tauschii: Sds-page Anasupporting

confidence: 74%

Novel x-Type High-Molecular-Weight Glutenin Genes From Aegilops tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

Zhang

Wang

et al. 2008

Genetics

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“…Electrospray mass spectrometry (ESI‐MS) and matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS have in part contributed to the assessment of gluten protein heterogeneity as well as the characterization of gliadins and glutenins. Previous studies have shown that MALDI‐TOF MS may allow molecular weight determination of purified HMW‐GS, which falls within the experimental error of those expected from the gene sequence 7–14. ESI‐MS, already applied to study a recombinant α‐gliadin,15 has been proved less suitable for HMW‐GS analysis, because of its scarce ionization efficiency.…”

Section: Introductionmentioning

confidence: 98%

Proteomic‐based analytical approach for the characterization of glutenin subunits in durum wheat

Mamone¹,

S²,

Luccia³

et al. 2009

J. Mass Spectrom.

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One of the main objectives of wheat glutenin subunit (GS) analysis is the identification of protein components linked to wheat quality. The proteomic characterization of glutenin has to consider the relatively low levels of arginine and lysine residues and the close sequence similarity among the different groups of these subunits, which hinders or even prevents the identification of the GS. In this study, a proteomic approach has been applied to resolve the heterogeneity of wheat glutenin components. Proteins extracted from Triticum durum flour were first analyzed by two-dimensional gel electrophoresis, which greatly reduced glutenin complexity. The identity of each spot was confirmed by nano liquid chromatography tandem mass spectrometry analysis of tryptic peptides. In parallel, measurements of the high mass range by matrix-assisted laser desorption/ionization time-of-flight analysis allowed detection of the large tryptic peptides. Gathering all data from search engine interrogation, very high sequence coverage was obtained for high molecular weight GS, including Bx7 and By8, in agreement with the known genetic profile of durum wheat. In addition, a truncated form of By8, never detected before, was also found. Low molecular weight GS (LMW-GS) B-type was identified with reasonable sequence coverage, while a clear identification of LWM-GS C- and D-type was hindered by the incompleteness of the wheat DNA databases. This study represents the first comprehensive analysis of the glutenin proteome and provides a reliable method for classifying wheat varieties according to their glutenin profile.

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“…Recently, studies on the characterization of glutenins have greatly improved toward the understanding of structures and functions of HMW-GS. In particular, proteomic technology centered with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has rapidly developed rapidly and become a powerful tool for identifying storage proteins (Alberghina et al 2005;Kussmann et al 1997;Muccilli et al 2005). Some recent investigations have focused on the tryptic peptide mapping of high molecular weight glutenin subunits (Cozzolino et al 2001), but little work on measuring accurate molecular weights of the intact HMW-GS within the mixture of HMW subunits has been reported.…”

Section: Introductionmentioning

confidence: 99%

Diversity of Novel Glutenin Subunits in Bread Wheat (Triticum aestivum L.)

et al. 2009

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Glutenin is a major determinant of baking performance and viscoelasticity, which are responsible for high-quality bread with a light porous crumb structure of a well-leavened loaf. We analyzed the diversity of glutenin genes from six wheat cultivars (Korean cvs. Keumgang and Jinpum, Chinese cvs. China-108 and Yeonnon-78, and Japanese cvs. Norin-61 and Kantou-107). Glutenins contain two types of isoforms such as high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin fractions were extracted from wheat endosperm using Osborne solubility method. A total of 217 protein spots were separated on twodimensional gel electrophoresis with isoelectric focusing (wide range of pH 3-10). The proteins spots were subjected to tryptic digestion and identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry. HMW-GS (43 isoforms) and LMW-GS (seven isoforms) are directly responsible for producing high-quality bread and noodles. Likewise, all the seed storage proteins are digested to provide nutrients for the embryo during seed germination and seedling growth. We identified the diverse glutenin subunits in wheat cultivars and compared the gluten isoforms among different wheat cultivars according to quality. This work gives an insight on the quality improvement in wheat crop.

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Proteomics of gluten: mapping of the 1Bx7 glutenin subunit in Chinese Spring cultivar by matrix‐assisted laser desorption/ionization

Cited by 22 publications

References 27 publications

Novel x-Type High-Molecular-Weight Glutenin Genes From Aegilops tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

Novel x-Type High-Molecular-Weight Glutenin Genes From Aegilops tauschii and Their Implications on the Wheat Origin and Evolution Mechanism of Glu-D1-1 Proteins

Proteomic‐based analytical approach for the characterization of glutenin subunits in durum wheat

Diversity of Novel Glutenin Subunits in Bread Wheat (Triticum aestivum L.)

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