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Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.

ABSTRACT: We have investigated the plasma proteome by using 2D gel electrophoresis and MS from patients with severe acute respiratory syndrome (SARS). A complete proteomic analysis was performed on four patients with SARS in different time courses, and a total of 38 differential spots were selected for protein identification. Most of the proteins identified are acute phase proteins, and their presence represents the consequence of serial cascades initiated by SARS-coronavirus infection. There are several proteins that have never been identified in plasma before using 2D gel electrophoresis, among which peroxiredoxin II was chosen for further study by analyzing additional 20 plasma samples from patients with probable and suspected SARS and patients with fever, respectively. The results showed that the level of plasma peroxiredoxin II in patients with SARS is significantly high and could be secreted by T cells. Taken together, our findings indicate that active innate immune responses, along with the oxidation-associated injuries, may play a major role in the pathogenesis of SARS.

SUBMITTER: Chen JH

PROVIDER: S-EPMC535397 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Chen Jenn-Han JH Chang Yu-Wang YW Yao Chen-Wen CW Chiueh Tzong-Shi TS Huang Su-Chin SC Chien Ko-Yi KY Chen An A Chang Feng-Yee FY Wong Chi-Huey CH Chen Yu-Ju YJ

Proceedings of the National Academy of Sciences of the United States of America 20041130 49

We have investigated the plasma proteome by using 2D gel electrophoresis and MS from patients with severe acute respiratory syndrome (SARS). A complete proteomic analysis was performed on four patients with SARS in different time courses, and a total of 38 differential spots were selected for protein identification. Most of the proteins identified are acute phase proteins, and their presence represents the consequence of serial cascades initiated by SARS-coronavirus infection. There are several ...[more]

PMID: 15572443

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Project description:BackgroundCertain 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.ResultsFlour 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.ConclusionsThis 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.

Project description:BackgroundWheat 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.ResultsGrain 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%.ConclusionsThis 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.

Dataset Information

Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Publications

Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry.

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