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PubMed2003

Bioverse: Functional, structural and contextual annotation of proteins and proteomes.

Functional annotation is routinely performed for large-scale genomics projects and databases. Researchers working on more specific problems, for instance on an individual pathway or complex, also need to be able to quickly, completely and accurately annotate sequences. The Bioverse sequence annotation server (http://bioverse.compbio.washington.edu) provides a web-based interface to allow users to submit protein sequences to the Bioverse framework. Sequences are functionally and structurally annotated and potential contextual annotations are provided. Researchers can also submit candidate genomes for annotation of all proteins encoded by the genome (proteome).

Internet↗
PubMed2004

Bioinformatics as a critical prerequisite to transcriptome and proteome studies.

Large-scale genomic studies rely strongly on annotations available in databases to design experimental supports such as arrays or to explain results in term of biological meaning. Most of this information originates from bioinformatic predictions. Their accuracy as well as their relevance to existing biological data are critical in avoiding the misinterpretation of experimental results.

Computational Biology↗
PubMed2006

NERVE: new enhanced reverse vaccinology environment.

BACKGROUND: Since a milestone work on Neisseria meningitidis B, Reverse Vaccinology has strongly enhanced the identification of vaccine candidates by replacing several experimental tasks using in silico prediction steps. These steps have allowed scientists to face the selection of antigens from the predicted proteome of pathogens, for which cell culture is difficult or impossible, saving time and money. However, this good example of bioinformatics-driven immunology can be further developed by improving in silico steps and implementing biologist-friendly tools. RESULTS: We introduce NERVE (New Enhanced Reverse Vaccinology Environment), an user-friendly software environment for the in silico identification of the best vaccine candidates from whole proteomes of bacterial pathogens. The software integrates multiple robust and well-known algorithms for protein analysis and comparison. Vaccine candidates are ranked and presented in a html table showing relevant information and links to corresponding primary data. Information concerning all proteins of the analyzed proteome is not deleted along selection steps but rather flows into an SQL database for further mining and analyses. CONCLUSION: After learning from recent years' works in this field and analysing a large dataset, NERVE has been implemented and tuned as the first available tool able to rank a restricted pool (approximately 8-9% of the whole proteome) of vaccine candidates and to show high recall (approximately 75-80%) of known protective antigens. These vaccine candidates are required to be "safe" (taking into account autoimmunity risk) and "easy" for further experimental, high-throughput screening (avoiding possibly not soluble antigens). NERVE is expected to help save time and money in vaccine design and is available as an additional file with this manuscript; updated versions will be available at http://www.bio.unipd.it/molbinfo.

Algorithms↗
PubMed2006

Piecing together a ciliome.

Cilia are slender microtubule-based appendages that emanate from the surfaces of a large proportion of eukaryotic cells. The motile and non-motile forms of cilia represent bona fide organelles comprising distinct repertoires of proteins that serve specific roles in locomotion or fluid movement, and sense chemical or physical extracellular cues. Owing in part to the growing number of genes associated with ciliary disorders, such as polycystic kidney disease and Bardet-Biedl syndrome, there has been a recent profusion of studies aimed at unveiling the protein makeup of cilia. The approaches used are complementary, involving several different organisms and spanning the fields of bioinformatics, genomics and proteomics. Here we review these studies and assess the various data sets to help define a comprehensive ciliary proteome, or 'ciliome'. We have compiled a cilia protein database that includes known cilia-associated proteins and numerous putative ciliary proteins including RAB-like small GTPases, which might be implicated in vesicular trafficking, and the microtubule-binding protein MIP-T3, some of which might be associated with ciliopathies.

Animals↗
PubMed2006

Proteome profiling in the rat Harderian gland.

The Harderian gland is an orbital gland located behind the ocular bulb in most terrestrial vertebrates probably functioning for production of lipid secretion to protect the eye. We herein present a protein reference database of the rat Harderian gland that may serve as analytical tool for future proteomic work, report lipid and porphyrin handling cascades, address sequence conflicts and report structures that have not been so far described by proteomics methods.

Amino Acid Sequence↗
PubMed2004

GELBANK: a database of annotated two-dimensional gel electrophoresis patterns of biological systems with completed genomes.

GELBANK is a publicly available database of two-dimensional gel electrophoresis (2DE) gel patterns of proteomes from organisms with known genome information (available at http://gelbank.anl.gov and ftp://bioinformatics.anl.gov/gelbank/). Currently it includes 131 completed, mostly microbial proteomes available from the National Center for Biotechnology Information. A web interface allows the upload of 2D gel patterns and their annotation for registered users. The images are organized by species, tissue type, separation method, sample type and staining method. The database can be queried based on protein or 2DE-pattern attributes. A web interface allows registered users to assign molecular weight and pH gradient profiles to their own 2D gel patterns as well as to link protein identifications to a given spot on the pattern. The website presents all of the submitted 2D gel patterns where the end-user can dynamically display the images or parts of images along with molecular weight, pH profile information and linked protein identification. A collection of images can be selected for the creation of animations from which the user can select sub-regions of interest and unlimited 2D gel patterns for visualization. The website currently presents 233 identifications for 81 gel patterns for Homo sapiens, Methanococcus jannaschii, Pyro coccus furiosus, Shewanella oneidensis, Escherichia coli and Deinococcus radiodurans.

Databases, Factual↗
PubMed2005

Data management and preliminary data analysis in the pilot phase of the HUPO Plasma Proteome Project.

The pilot phase of the HUPO Plasma Proteome Project (PPP) is an international collaboration to catalog the protein composition of human blood plasma and serum by analyzing standardized aliquots of reference serum and plasma specimens using a variety of experimental techniques. Data management for this project included collection, integration, analysis, and dissemination of findings from participating organizations world-wide. Accomplishing this task required a communication and coordination infrastructure specific enough to support meaningful integration of results from all participants, but flexible enough to react to changing requirements and new insights gained during the course of the project and to allow participants with varying informatics capabilities to contribute. Challenges included integrating heterogeneous data, reducing redundant information to minimal identification sets, and data annotation. Our data integration workflow assembles a minimal and representative set of protein identifications, which account for the contributed data. It accommodates incomplete concordance of results from different laboratories, ambiguity and redundancy in contributed identifications, and redundancy in the protein sequence databases. Recommendations of the PPP for future large-scale proteomics endeavors are described.

Algorithms↗
PubMed2002

Primer on medical genomics. Part IV: Expression proteomics.

Proteomics, simply defined, is the study of proteomes. More completely, proteomics is defined as the study of all proteins, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, in a given cell or organism within a given environment and at a specific stage in the cell cycle. Proteins carry out the biological functions encoded by genes; hence, once the initial stage of genome sequencing and gene discovery is completed, a study of the proteome must be undertaken to address fundamental biological questions. The 3 broad areas are expression proteomics, which catalogues the relative abundance of proteins; cell-mapping or cellular proteomics, which delineates functional protein-protein interactions and organelle-specific protein distribution; and structural proteomics, which characterizes the 3-dimensional structure of proteins. With these approaches, proteins are studied on a global scale using a synergistic combination of powerful, high-throughput technologies, including 2-dimensional polyacrylamide gel electrophoresis, mass spectrometry, multidimensional liquid chromatography, and bioinformatics. Mass spectrometry, which provides highly accurate molecular mass measurements, has emerged as the analytical technology of choice for protein identification, characterization, and sequencing. This task has been made considerably easier with the availability of complete, nonredundant, and annotated genome sequence databases for many organisms. This article reviews the area of expression proteomics.

Biotechnology↗
PubMed2005

Comparative proteomic analysis of peripheral blood eosinophils from healthy donors and atopic dermatitis patients with eosinophilia.

Atopic dermatitis (AD) is an allergic disease that has recently shown a dramatic increase of incidence in developed countries. Eosinophilia, the accumulation of eosinophils, occurs in AD patients through an anti-apoptotic mechanism. To understand the target proteins involved in the anti-apoptotic signaling of eosinophilia, we used a proteomic approach to analyze eosinophil proteins from AD patients with eosinophilia and healthy donors. Protein spots in two-dimensional electrophoresis (2-DE) gels were identified with peptide mass fingerprinting (PMF) based on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and database searching. More spots were observed in the 2-DE proteome map from AD patient samples (1310 +/- 58 spots) than in those from healthy donors (1121 +/- 40 spots). We identified 51 proteins affected by eosinophilia: 19 related to signaling, 8 involved in regulation of metabolism, 4 related to apoptosis, and 3 involved in inflammation. The other identified proteins were associated with transcription, RNA processing, translation, the cytoskeleton, and unknown functions. Among the identified proteins, we observed prominent increases in the expressions of cyclinA2, voltage-dependent anion channel protein 2, and 38 kDa FK506 binding protein 8 in eosinophils from AD patients in comparison to healthy donors. PMF and immunoblotting of a single spot that was expressed in eosinophils from healthy individuals but not in AD patients identified the protein as phosphorylated growth receptor binding 7 (Grb7) adaptor protein. Increased phosphorylation of Grb7 and its upstream signaling protein, focal adhesion kinase (FAK), was detected in low viability eosinophils such as those from healthy donors or in cultured eosinophils (AML14.3D10 cells) treated with dexamethasone. These results suggest that phosphorylation of Grb7 and the expressions of cyclinA2, voltage-dependent anion channel protein 2, and 38 kDa FK506 binding protein 8 may be related with the anti-apoptosis mechanism of eosinophilia.

Apoptosis↗
PubMed2003

OLAV: towards high-throughput tandem mass spectrometry data identification.

Mass spectrometry combined with database searching has become the preferred method for identifying proteins in proteomics projects. Proteins are digested by one or several enzymes to obtain peptides, which are analyzed by mass spectrometry. We introduce a new family of scoring schemes, named OLAV, aimed at identifying peptides in a database from their tandem mass spectra. OLAV scoring schemes are based on signal detection theory, and exploit mass spectrometry information more extensively than previously existing schemes. We also introduce a new concept of structural matching that uses pattern detection methods to better separate true from false positives. We show the superiority of OLAV scoring schemes compared to MASCOT, a widely used identification program. We believe that this work introduces a new way of designing scoring schemes that are especially adapted to high-throughput projects such as GeneProt large-scale human plasma project, where it is impractical to check all identifications manually.

Bayes Theorem↗
PubMed2003

Linking genotype to phenotype: the International Rice Information System (IRIS).

The International Rice Information System (IRIS, http://www.iris.irri.org) is the rice implementation of the International Crop Information System (ICIS, http://www.icis.cgiar.org), a database system for the management and integration of global information on genetic resources and germplasm improvement for any crop. Building upon the germplasm genealogy and field data components of ICIS, IRIS is being extended to handle diverse rice genomics data including: genetic mapping, genome annotation, genotype, mutant, transcripteome, proteome and metabolomic data. Users can access information in the database through stand-alone programs and WWW interfaces offering specialist views to researchers with different interests.

Database Management Systems↗
PubMed2003

Proteomics in Alzheimer's disease: insights into potential mechanisms of neurodegeneration.

Proteomics involves the identification of unknown proteins following their separation, often using two-dimensional electrophoresis, digestion of particular proteins of interest by trypsin, determination of the molecular weight of the resulting peptides, and database searching to make the identification of the proteins. Application of proteomics to Alzheimer's disease (AD), the major dementing disorder of the elderly, has just begun. Differences in protein expression and post-translational modification (mostly oxidative modification) of proteins from AD brain and peripheral tissue, as well as in brain from rodent models of AD, have yielded insights into potential molecular mechanisms of neurodegeneration in this dementing disorder. This review surveys the proteomics studies relevant to AD, from which new understandings of the pathology, biochemistry, and physiology of AD are beginning to emerge.

Alzheimer Disease↗
PubMed2002

Functional genomics of the social amoebae, Dictyostelium discoideum.

Dictyostelium discoideum is one of the simplest organisms to form a multicellular structure, and it offers several advantages as a model. In order to understand the genetic basis of the multicellular development, a comprehensive analysis of cDNAs is being performed. To date, about 75,000 ESTs have been collected at different stages of development. They have been assembled into about 6,400 independent sequences that represent 70-80% of all of the expected genes in D. discoideum. The results are available on the Internet. In addition to structural analyses, functional analyses of the temporal and spatial expression patterns and gene targeting are being carried out. Furthermore, there are plans to combine the information that is obtained from the cDNA, Genome, and Proteome Projects, as well as the published results, into an integrated database, DictyBase.

Animals↗
PubMed1999

Proteome analysis. II. Protein subcellular redistribution: linking physiology to genomics via the proteome and separation technologies involved.

While annotated two-dimensional (2D) gel electrophoresis databases contain thousands of proteins, they do not represent the entire genome. High-molecular-mass proteins in particular are conspicuously absent from such databases. Filamin is prototypical of this class of proteins since it is a dimer with relative molecular mass (Mr) of 520000 containing at least 240 potential phosphorylation sites. Filamin is not readily separated by current 2D procedures, and is difficult to study with respect to cycles of phosphorylation-dephosphorylation. Novel technologies are needed to identify biochemical pathways impinging upon such targets. The success of immunofluorescence microscopy as a research tool can be attributed in part to the fact that proteins redistribute in response to a variety of physiological stimuli. Comparable quantitative methods are required in proteome analysis. Three components are necessary for development of an approach that is capable of screening for protein redistribution events: (1) subcellular fractionation, (2) protein labeling and (3) data acquisition. An integrated approach is presented that utilizes differential detergent fractionation combined with reversible, luminescent protein stains and analytical imaging for high-throughput analysis of signal transduction events leading to protein subcellular redistribution. The procedure has been successfully implemented to rapidly define key second messenger pathways leading to endothelial cell junctional permeability and to guide in the design of a new family of peptide-based anti-inflammatory drugs.

Amino Acid Sequence↗
PubMed2006

Quantitative proteomic analysis of the budding yeast cell cycle using acid-cleavable isotope-coded affinity tag reagents.

Quantitative profiling of proteins, the direct effectors of nearly all biological functions, will undoubtedly complement technologies for the measurement of mRNA. Systematic proteomic measurement of the cell cycle is now possible by using stable isotopic labeling with isotope-coded affinity tag reagents and software tools for high-throughput analysis of LC-MS/MS data. We provide here the first such study achieving quantitative, global proteomic measurement of a time-course gene expression experiment in a model eukaryote, the budding yeast Saccharomyces cerevisiae, during the cell cycle. We sampled 48% of all predicted ORFs, and provide the data, including identifications, quantitations, and statistical measures of certainty, to the community in a sortable matrix. We do not detect significant concordance in the dynamics of the system over the time-course tested between our proteomic measurements and microarray measures collected from similarly treated yeast cultures. Our proteomic dataset therefore provides a necessary and complementary measure of eukaryotic gene expression, establishes a rich database for the functional analysis of S. cerevisiae proteins, and will enable further development of technologies for global proteomic analysis of higher eukaryotes.

Carbon Isotopes↗
PubMed2004

Tackling the plant proteome: practical approaches, hurdles and experimental tools.

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.

Electrophoresis, Gel, Two-Dimensional↗