Search

We have identified a prominent ~10-kDa protein biomarker observed in the matrix-assisted laser desorption/ ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobaeter: jejuni, coli, lari, upsaliensis, and helvetieus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari (2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a ~10.5-kDa protein biomarker of a C. concisus strain as an HU protein.

Abstract Background Mineral nutrition during wheat grain development has large effects on wheat flour protein content and composition, which in turn affect flour quality and immunogenic potential for a commodity of great economic value. However, it has been difficult to define the precise effects of mineral nutrition on protein composition because of the complexity of the wheat flour proteome. Recent improvements in the identification of flour proteins by tandem mass spectrometry (MS/MS) and the availability of a comprehensive proteome map of flour from the US wheat Butte 86 now make it possible to document changes in the proportions of individual flour proteins that result from the application of mineral nutrition. Results Plants of Triticum aestivum 'Butte 86' were grown with or without post-anthesis fertilization (PAF) and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. Significant changes in the proportions of 54 unique proteins were observed as a result of the treatment. Most omega-gliadins, high molecular weight glutenin subunits (HMW-GS) and serpins as well as some alpha-gliadins increased in proportion with PAF. In contrast, alpha-amylase/protease inhibitors, farinins, purinins and puroindolines decreased in proportion. Decreases were also observed in several low molecular weight glutenin subunits (LMW-GS), globulins, defense proteins and enzymes. The ratio of HMW-GS to LMW-GS in the flour increased from 0.61 to 0.95 and the ratio of gliadins to glutenins increased from 1.02 to 1.30 with PAF. Because flour protein content doubled with PAF from 7 to 14%, most protein types actually increased in absolute amount (μg/mg flour protein). Data further suggest that flour proteins change with PAF according to their content of sulfur-containing amino acids Cys + Met. Conclusions A 2-DE approach revealed changes in the wheat flour proteome due to PAF that are important for flour quality and immunogenic potential. The work forms a baseline for further studies of the effects of environmental variables on flour protein composition and provides clues about the regulation of specific flour protein genes. The study also is important for identifying targets for breeding programs and biotechnology efforts aimed at improving flour quality.

Abstract Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants.

Abstract Background Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. Results Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. Conclusions This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression.

Abstract 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.

A sensitive method to quantitate the methionine sulfoxide present at position 213 (MetSO213) is developed to measure the changes in MetSO213 over the time course of an intracranial challenge, using the 263K strain of hamster-adapted scrapie. The data indicated that MetSO213 is not found to be a prion-specific covalent signature and the proportion of Met213 that is oxidized decreases over the course of the disease.

Wheat gliadins are a complex group of proteins that contribute to the functional properties of wheat flour doughs and contain epitopes that are relevant for celiac disease (CD) and wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, we extracted ethanol-soluble gliadin fractions from flour of the Korean bread wheat cultivar Keumkang. Proteins were separated by 2-dimensional gel electrophoresis (2-DE) using a pI range of 6–11 in the first dimension and subjected to tandem mass spectrometry. α-, γ-, and ω-gliadins were identified as the predominant proteins in 31, 28, and one 2-DE spot, respectively. An additional six ω-gliadins were identified in a separate experiment in which a pI range of 3–11 was used for protein separation. We analyzed the composition of CD- and WDEIA-relevant epitopes in the gliadin sequences from Keumkang flour, demonstrating the immunogenic potential of this cultivar. Detailed knowledge about the complement of gliadins accumulated in Keumkang flour provides the background necessary to devise either breeding or biotechnology strategies to improve the functional properties and reduce the adverse health effects of the flour. [ABSTRACT FROM AUTHOR]

Background: Citrus represents a crop of global importance both in economic impact and significance to nutrition. Citrus production worldwide is threatened by the disease Huanglongbing (HLB), caused by the phloem-limited pathogen Liberibacter spp.. As a source of stable HLB-resistance has yet to be identified, there is Candidatus considerable interest in characterization of novel disease-associated citrus genes. Results: A gene family of Small Cyclic Amphipathic Peptides (SCAmpPs) in citrus is described. The citrus genomes contain 100-150 SCAmpPs genes, approximately 50 of which are represented in the citrus EST database. These genes encode small ~50 residue precursor proteins that are post-translationally processed, releasing 5-10 residue cyclic peptides. The structures of the SCAmpPs genes are highly conserved, with the small coding domains interrupted by a single intron and relatively extended untranslated regions. Some family members are very highly transcribed in specific citrus tissues, as determined by representation in tissue-specific cDNA libraries. Comparison of the ESTs of related SCAmpPs revealed an unexpected evolutionary profile, consistent with targeted mutagenesis of the predicted cyclic peptide domain. The SCAmpPs genes are displayed in clusters on the citrus chromosomes, with apparent association with receptor leucine-rich repeat protein arrays. This study focused on three SCAmpPs family members with high constitutive expression in citrus phloem. Unexpectedly high sequence conservation was observed in the promoter region of two phloem-expressed SCAmpPs that encode very distinct predicted cyclic products. The processed cyclic product of one of these phloem SCAmpPs was characterized by LC-MS-MS analysis of phloem tissue, revealing properties consistent with a K+ ionophore. Conclusions: The SCAmpPs amino acid composition, protein structure, expression patterns, evolutionary profile and chromosomal distribution are consistent with designation as ribosomally synthesized defense-related peptides. [ABSTRACT FROM AUTHOR]

Background: Wheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain. It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase. Results: We performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene wasmutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene. Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing. Conclusions: Our results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence. [ABSTRACT FROM AUTHOR]

Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data. [ABSTRACT FROM AUTHOR]

Abstract: Genetic transformation via the biolistic method has been used to introduce genes encoding natural and novel high-molecular-weight glutenin subunits (HMW-GS) into wheat. The appearance of new seed proteins of sizes not predicted by the transgene coding sequences was noted in some experiments. In this report, the identities of thirteen of these novel proteins were determined by tandem mass spectrometry (MS/MS). Seven different proteins larger than and two proteins smaller than the native protein were shown to contain peptides from 1Dx5. A novel protein found in some progeny of crosses between a transgenic plant and Great Plains winter wheats was larger than but contained several peptides from 1Dy10. In one line, a protein larger than and a protein smaller than HMW-GS each contained peptides from the N- and C-terminus of 1Dx5 and from the repeat region of 1Dy10. In a sixth transgenic line, the native Bx7 gene was apparently replaced by a gene that encodes a larger version of 1Bx7. The variant proteins accumulate in the polymeric protein fraction, indicating that they can form inter-molecular disulfide bonds. These results show that novel proteins found in some transformants are encoded by altered versions of either the transforming or endogenous HMW-GS genes. [Copyright &y& Elsevier]

Background: Flour quality is largely determined by the gluten proteins, a complex mixture of proteins consisting of high molecular weight-glutenin subunits (HMW-GS), low molecular weight-glutenin subunits (LMW-GS), and a-, ?-, and ?-gliadins. Detailed proteomic analyses of the effects of fertilizer and high temperature on individual gliadin and glutenin protein levels are needed to determine how these environmental factors influence flour quality. Results: Wheat plants (Triticum aestivum L. cv. Butte 86) were grown in greenhouses under moderate and high temperature regimens with and without post-anthesis fertilizer. Quantitative two-dimensional gel electrophoresis was used to construct accumulation profiles in developing endosperm for the entire complement of gluten proteins identified previously by tandem mass spectrometry. Amounts of individual gliadins and glutenins were also determined in flour produced under each of the regimens. Under all environmental regimens, most HMW-GS, LMW-GS, ?- and ?-gliadins accumulated rapidly during early stages of grain development and leveled off during middle stages of development. A subset of LMW-GS showed a second distinct profile, accumulating throughout development, while a-gliadins showed a variety of accumulation profiles. In flour, fourteen distinct gluten proteins responded similarly to fertilizer, high temperature, and high temperature plus fertilizer. The majority of HMW-GS and ?-gliadins and some a-gliadins increased while two LMW-GS and a minor ?-gliadin decreased. Fertilizer did not influence gluten protein accumulation under high temperature conditions. Additionally, the effects of fertilizer and high temperature were not additive; very few changes were observed when plants that received fertilizer were subjected to high temperature. Conclusions: Although post-anthesis temperature and fertilizer have very different effects on grain development and yield, the two treatments elicit surprisingly similar effects on the accumulation of gluten proteins. The similarity of the responses to the different treatments is likely due to source-sink activities of nitrogen reserves in the wheat plant. Because each protein that showed a response in this study is linked to a gene sequence, the work sets the stage for transgenic studies that will better elucidate the roles of specific proteins in flour quality and in the response to the environment. [ABSTRACT FROM AUTHOR]

Background: Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. Results: Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alphagliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. Conclusions: This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression. [ABSTRACT FROM AUTHOR]

Abstract: The complement of alpha gliadins expressed in a single wheat cultivar was examined by assembling expressed sequence tags (ESTs) from Triticum aestivum cv. Butte 86. Twelve of 19 resulting contigs encoded complete proteins, but only two were identical to proteins reported previously. Eight contained various combinations of epitopes important in celiac disease (CD), while four lacked typical CD epitopes. One alpha gliadin contained an additional cysteine residue that could allow incorporation of the protein into glutenin polymers. In addition, two new types of alpha gliadins ending in GFFGTN and GIMSTN were identified. Based on the number of ESTs, genes encoding proteins that contained the greatest numbers of CD epitopes were expressed at the highest levels. Proteins corresponding to 12 contigs were distinguished in Butte 86 flour by tandem mass spectrometry (MS/MS). Unique peptide tags for individual alpha gliadins, including some that lack CD epitopes and the protein containing the extra cysteine, are reported. The ability to distinguish closely related alpha gliadins by MS/MS makes it possible to explore the roles of individual proteins in flour quality, better define relationships between specific proteins and celiac disease, and devise strategies to reduce the immunogenic potential of wheat flour for patients with CD. [Copyright &y& Elsevier]

Abstract: The accumulation of KCl-soluble/methanol-insoluble albumins and globulins was investigated in the endosperm of developing wheat (Triticum aestivum, L. cv. Butte 86) grain produced under a moderate (24°C/17°C, day/night) or a high temperature regimen (37°C/28°C) imposed from 10 or 20 days post-anthesis (dpa) until maturity. Proteins were separated by 2-DE and developmental profiles for nearly 200 proteins were analyzed by hierarchical clustering. Comparison of protein profiles across physiologically equivalent stages of grain fill revealed that high temperature shortened, but did not substantially alter, the developmental program. Accumulation of proteins shifted from those active in biosynthesis and metabolism to those with roles in storage and protection against biotic and abiotic stresses. Few proteins responded transiently when plants were transferred to the high temperature regimens, but levels of a number of proteins were altered during late stages of grain development. Specific protein responses depended on whether the high temperature regimens were initiated early or mid development. Some of the heat responsive proteins have been implicated in gas bubble stabilization in bread dough and others are suspected food allergens. [Copyright &y& Elsevier]

Application of a thiol-specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin-linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed. [Copyright &y& Elsevier]

Abstract: Effects of mineral nutrition and temperature on accumulation and composition of protein in wheat grains and on baking quality were studied under controlled environments. Under a moderate temperature regimen of 24°C days and 17°C nights (24/17°C), post-anthesis N:P:K 20:20:20 (NPK) supplied by continuous drip irrigation increased the rate of protein accumulation, doubled flour protein percentage and slightly increased final single kernel weight. In contrast, post-anthesis NPK had almost no effect on rate or duration of protein accumulation or flour protein percentage under a high temperature regimen of 37°C days and 28°C nights (37/28°C). The 37/28°C regimen shortened and compressed the stages of grain fill, reduced the duration of dry matter accumulation, and reduced single kernel weight by 50%. Rate and duration of protein accumulation in thermal time and total protein per grain at 37/28°C with or without NPK were similar to that at 24/17°C in the absence of post-anthesis NPK. Protein percentage was higher for flour from grain produced at 37/28°C with or without NPK than at 24/17°C in the presence of post-anthesis NPK. Transcript and protein profiling studies confirmed that the 37/28°C regimen compressed development without disrupting coordinate synthesis of gliadins and glutenin subunits, although some specific effects of NPK and temperature on relative amounts of individual gliadins and glutenins were observed. Transcript levels for ω-gliadins, α-gliadins and high molecular weight glutenin subunits (HMW-GS) declined at 24/17°C in the absence of post-anthesis NPK, whereas transcript levels for low molecular weight glutenin subunits (LMW-GS) and γ-gliadins showed little change. Two-dimensional gel electrophoresis (2DE) demonstrated that relative spot volumes for several ω-gliadins, α-gliadins and HMW-GS were lower at 24/17°C in the absence than in the presence of post-anthesis NPK, whereas the relative spot volume for a major LMW-GS was lower in the presence of NPK. Effects of temperature on relative spot volume were generally smaller than effects of NPK. Compared to the 24/17°C regimen in the absence of post-anthesis NPK, relative spot volume for some α-gliadins and HMW-GS were higher at 37/28°C, with or without NPK, and relative spot volume for a major LMW-GS decreased at 37/28°C. Loaf volume was correlated with flour protein percentage regardless of temperature regimen but mixing tolerance was highest for flour from grain produced under the 24/17°C regimens with NPK. [Copyright &y& Elsevier]

Both genetic and environmental factors influence the types and amounts of wheat proteins that link together to form polymers essential for flour quality. To understand how plant growth conditions might influence gluten polymer formation, protein fractions containing small and large polymers were separated from flour from the US wheat Butte 86 grown in the absence or presence of post-anthesis fertilizer. Proteins in the polymer fractions were analyzed by quantitative two-dimensional gel electrophoresis (2-DE). The ratio of high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS) increased in both fractions in response to fertilizer, due in part to small increases in the proportions of individual HMW-GS. There were also changes within the LMW-GS. In particular, omega and alpha chain terminators increased in proportion in both polymer fractions, but changes were more pronounced in the large polymer fractions. Serpins also increased in both polymer fractions. Additionally, the study revealed differences in the proportions of traditional LMW-GS in small and large polymer fractions. LMW-s type proteins were more abundant in the large polymers while LMW-i type proteins were more prevalent in the small polymers, suggesting that these proteins may play different roles in the gluten polymer. [ABSTRACT FROM AUTHOR]

Abstract: Although a principal source of energy and protein for millions of the world''s poorest people, the nutritional value of sorghum (Sorghum bicolor L. Moench) is diminished because of low digestibility of grain protein and starch. To address this problem, we analyzed the properties of two sorghum lines that have a common pedigree but differ in digestibility. Consistent with results based on a ruminal fluid assay, the protein and starch of one line (KS48) was more thoroughly digested than that of the other (KS51) using in vitro assays based on pepsin and α-amylase. The indigestibility of KS51 relative to KS48 was shown to be due to (i) a greater abundance of disulfide-bonded proteins; (ii) presence in KS51 of non-waxy starch and the accompanying granule-bound starch synthase; and (iii) the differing nature of the protein matrix and its interaction with starch. The current findings suggest that each of these factors should be considered in efforts to enhance the nutritional value of sorghum grain. [Copyright &y& Elsevier]

Abstract: Twenty-three expressed sequence tags (ESTs) from the US spring wheat Butte 86 were identified that encode proteins similar to a globulin-2 protein from maize embryos. The ESTs assembled into three contigs, two of which include the entire coding region for the mature protein. The encoded proteins contain two cupin domains and show significant identities with 7S seed proteins from other species that are known or putative food allergens. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) with primers specific for two of the sequences demonstrated that the globulin-2 genes are expressed late in grain development and that transcript levels increase when grain is produced under high temperature conditions. Transcripts were detected in both whole grain and endosperm, but levels were significantly higher in whole grain and highest in embryo. In wheat flour, at least 17 protein spots that differ in both size and pI were identified as globulin-2 by 2-DE/MS. Seven of the spots increased more than 2-fold in relative proportion when grain was produced under high temperature regimens. The data suggest that both transcriptional and post-translational mechanisms are involved in the response of globulin-2 to high temperatures. [Copyright &y& Elsevier]

Abstract: Genetic variability of polyphenol oxidase (PPO) activity was evaluated in hexaploid, tetraploid, and aneuploid wheat (Triticum spp.). A DNA probe to kernel-specific PPO hybridized to PPO clones from an immature wheat kernel cDNA library but not to clones from a seedling cDNA library. Recombinant protein and antibody were developed from the kernel-specific PPO. PPO transcript and antigenic proteins were most abundant during kernel development and decreased rapidly at physiological maturity. PPO proteins were more abundant in immature kernels, but PPO activity was greater in mature kernels. Wheat kernel antigenic protein bands of ∼58, 60, 62, and 75kD were identified; immunoprecipitated proteins of ∼60 and ∼62kD were confirmed to be PPOs by LC-MS/MS. The 2D chromosome was implicated as a major locus of PPO activity in cultivar (cv.) ‘Chinese Spring’ based on antigenic staining intensity of ∼60–62kD proteins and kernel PPO activity in aneuploid lines. The ∼58kD protein was not well-correlated with kernel PPO activity, while the ∼75kD protein was likely a PPO precursor. Tropolone completely inhibited PPO activity extracted from mature kernels but not from immature kernels. Total PPO activity in mature kernels is thus a function of specific PPO isoforms present, their abundance, and activation levels. [Copyright &y& Elsevier]

We have identified a prominent ∼10-kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lad, upsaliensis, and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari (2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MAIDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a ∼10.5-kDa protein biomarker of a C. concisus strain as an HU protein. [ABSTRACT FROM AUTHOR]

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm. [Copyright &y& Elsevier]