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Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gam

Analysis of expressed sequence tags from a single wheat cultivar facilitates interpretation of tandem mass spectrometry data and discrimination of gamma gliadin proteins that may play different functional roles in flour   (Citations: 4)
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BACKGROUND: The gamma gliadins are a complex group of proteins that together with other gluten proteins determine the functional properties of wheat flour. The proteins have unusually high levels of glutamine and proline and contain large regions of repetitive sequences. While most gamma gliadins are monomeric proteins containing eight conserved cysteine residues, some contain an additional cysteine residue that enables them to be linked with other gluten proteins into large polymers that are critical for flour quality. The ability to differentiate among the gamma gliadins is important for studies of wheat flour quality because proteins with similar sequences can have different effects on functional properties. RESULTS: The complement of gamma gliadin genes expressed in the wheat cultivar Butte 86 was evaluated by analyzing publicly available expressed sequence tag (EST) data. Eleven contigs were assembled from 153 Butte 86 ESTs. Nine of the contigs encoded full-length proteins and four of the proteins contained nine cysteine residues. Only one of the encoded proteins was a perfect match with a sequence reported in NCBI. Contigs from four different publicly available EST assemblies encoded proteins that were perfect matches with some, but not all, of the Butte 86 gamma gliadins and the complement of identical proteins was different for each assembly. A specialized database that included the sequences of Butte 86 gamma gliadins was constructed for identification of flour proteins by tandem mass spectrometry (MS/MS). In a pilot experiment, proteins corresponding to six Butte 86 gamma gliadin contigs were distinguished by MS/MS, including one containing the extra cysteine residue. Two other proteins were identified as one of two closely related Butte 86 proteins but could not be distinguished unequivocally. Unique peptide tags specific for Butte 86 gamma gliadins are reported. CONCLUSIONS: Inclusion of cultivar-specific gamma gliadin sequences in databases maximizes the number and quality of peptide identifications and increases sequence coverage of these gamma gliadins by MS/MS. This approach makes it possible to distinguish closely related proteins, to associate individual proteins with sequences of specific genes, and to evaluate proteomic data in a biological context to better address questions about wheat flour quality.
Journal: BMC Plant Biology - BMC PLANT BIOL , vol. 10, no. 1, pp. 7-14, 2010
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    • ...In addition, some proteins with gliadin-like sequences have an odd number of Cys residues and can be linked to the glutenin polymer [7,12-16]...
    • ...Although over one million wheat ESTs have been arranged into provisional contigs that combine sequences from multiple cultivars, they do not represent the exact sequences for proteins of a single variety [12,13]...
    • ...Key to this approach was conducting a two pass database search, first against a large set of protein sequences and then against a subset database that also contained decoy sequences [12,13,57-59]...
    • ...Flour was obtained from the US hard red spring wheat Butte 86. Genes within several complex families of grain proteins have been characterized in detail in this cultivar [12,13,41,51]...
    • ...Therefore a final manual evaluation of the peptide assignments was carried out as in [12,13]...
    • ... one gamma-gliadin type and 12 spots contained one or more additional protein types, such as traces of an alpha-gliadin or LMW-GS (Additional file 1). Scaffold assigned peptides to 13 gamma-gliadin sequences, including two from NCBI nr, one from a large contig assembly and ten from Butte 86 contigs (Table 2). After manual analysis, all peptides were found to match partial (2) or complete (7) gammagliadin contigs assembled for Butte 86 [12] ...
    • ...Previously through EST analysis, we identified coding sequences for twelve full-length and seven partial alpha-gliadins as well as nine full-length and two partial gamma-gliadin sequences expressed in Butte 86 [12,13]...
    • ...Value of cultivar-specific databases Rapid evolution of the gluten proteins has led to minor differences in amino acid sequence that made it surprisingly difficult to use existing sequence databases for precise identification of gliadins and LMW-GS [10,12,13,31]...
    • ...Small differences, such as a few extra Gln in the repetitive region of a gliadin sequence or one amino acid change that altered a proteolytic site result in proteolytic or fragmentation patterns that are not recognized [12,13]...
    • ... [84], nucleotide sequences translated in all six reading frames of contigs from TaGI Releases 10.0 and 11.0 [63], US Wheat Genome Project [85], HarvEST 1.14 (WI all NSF “stringent” assembly from 05/08/04) [86], NCBI Unigene Build #55 [84], and all ESTs from Butte 86 developing grain, as well as translated sequences (reading frame only) of 94 Butte 86 contigs (Additional file 2), including those for alpha-gliadins and gamma-gliadins [ ...
    • ...In these cases, the MS/MS data from the spot was further inspected as described [12]...
    • ...Procedures to develop a set of contigs representing gamma-gliadins and alpha-gliadins for the variety Butte 86 [12,13] were also used to develop contig sets from Butte 86 ESTs that encoded LMW-GS, omega-gliadins, beta-amylases, farinins, purinins and globulins...

    Frances M Dupontet al. Deciphering the complexities of the wheat flour proteome using quantit...

    • ...ESTs were assembled with Lasergene Seqman Pro (DNASTAR, Inc., Madison, WI) using the Classic Assembler with default settings except that the minimum match percentage was set to 98 and the minimum sequence length was set to 50. Assemblies were inspected manually and mismatches that occurred in overlap regions of ESTs were resolved by examining phred quality scores for individual ESTs as detailed in Altenbach et al. [35]...

    Susan B Altenbachet al. The spectrum of low molecular weight alpha-amylase/protease inhibitor ...

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