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At least 811 records · Page 45Linked to original sources
PubMed2003

Investigating protein domain combinations in complete proteomes.

Protein-related information is more accumulated rather than reduced to a synthetic view. Itemising properties of protein sequences is informative, so is the list of ingredients to do some cooking, but without a recipe, that is, quantification and chronology, understanding is incomplete. If the goal of accumulating information is to discover or reveal the function and related biochemical mechanisms, information has to be weighed and ordered. As a guideline, the weight of a piece of information should reflect how often it consistently occurs in various contexts. We propose a common sense approach to quantify and put data and information into perspective. Complete bacterial proteomes are individually mapped with the Pfam-A database of domains and protein family signatures in an attempt to assess the modularity of proteins at the level of a single proteome and the implications of a modular description of proteins for a functional interpretation. Poorly annotated proteins in the most documented bacteria (E. coli and B. subtilis) were considered in an attempt to formulate hypothesis on the basis of domain/module content.

Bacillus subtilis↗
PubMed2004

A proteomic analysis of maize chloroplast biogenesis.

Proteomics studies to explore global patterns of protein expression in plant and green algal systems have proliferated within the past few years. Although most of these studies have involved mapping of the proteomes of various organs, tissues, cells, or organelles, comparative proteomics experiments have also led to the identification of proteins that change in abundance in various developmental or physiological contexts. Despite the growing use of proteomics in plant studies, questions of reproducibility have not generally been addressed, nor have quantitative methods been widely used, for example, to identify protein expression classes. In this report, we use the de-etiolation ("greening") of maize (Zea mays) chloroplasts as a model system to explore these questions, and we outline a reproducible protocol to identify changes in the plastid proteome that occur during the greening process using techniques of two-dimensional gel electrophoresis and mass spectrometry. We also evaluate hierarchical and nonhierarchical statistical methods to analyze the patterns of expression of 526 "high-quality," unique spots on the two-dimensional gels. We conclude that Adaptive Resonance Theory 2-a nonhierarchical, neural clustering technique that has not been previously applied to gene expression data-is a powerful technique for discriminating protein expression classes during greening. Our experiments provide a foundation for the use of proteomics in the design of experiments to address fundamental questions in plant physiology and molecular biology.

Chloroplasts↗
PubMed2003

ABRF-PRG03: phosphorylation site determination.

A fundamental aspect of proteomics is the analysis of post-translational modifications, of which phosphorylation is an important class. Numerous nonradioactivity-based methods have been described for high-sensitivity phosphorylation site mapping. The ABRF Proteomics Research Group has conducted a study to help determine how many laboratories are equipped to take on such projects, which methods they choose to apply, and how successful the laboratories are in implementing particular methodologies. The ABRF-PRG03 sample was distributed as a tryptic digest of a mixture of two proteins with two synthetic phosphopeptides added. Each sample contained 5 pmol of unphosphorylated protein digest, 1 pmol of each phosphopeptide from the same protein, and 200 fmol of a minor protein component. Study participants were challenged to identify the two proteins and the two phosphorylated peptides, and determine the site of phosphorylation in each peptide. Almost all respondents successfully identified the major protein component, whereas only 10% identified the minor protein component. Phosphorylation site analysis proved surprisingly difficult, with only 3 of the 54 laboratories correctly determining both sites of phosphorylation. Various strategies and instruments were applied to this task with mixed success; chromatographic separation of the peptides was clearly helpful, whereas enrichment by metal affinity chromatography met with surprisingly little success. We conclude that locating sites of phosphorylation remains a significant challenge at this level of sample abundance.

Chemistry Techniques, Analytical↗
PubMed2005

Decoding two-dimensional polyacrylamide gel electrophoresis complex maps by autocovariance function: a simplified approach useful for proteomics.

This paper describes a mathematical approach applied for decoding the complex signal of two-dimensional polyacrylamide gel electrophoresis maps of protein mixtures. The method is helpful in extracting analytical information since separation of all the proteins present in the sample is still far from being achieved and co-migrating proteins are generally present in the same spot. The simplified method described is based on the study of the 2-D autocovariance function (2D-ACVF) computed on an experimental digitized map. The first part of the 2D-ACVF allows for the estimation of the number of proteins present in the sample (2D-ACVF computed at the origin) and of the separation performance (mean spot size). Moreover, the 2D-ACVF plot is a powerful tool in identifying order in the spot position, and singling it out from the complex separation pattern. This method was validated on synthetic maps obtained by computer simulation to describe 2-D PAGE real maps and reference maps retrieved from the SWISS-2DPAGE database. The results obtained are discussed by focusing on specific information relevant in proteomics: sample complexity, separation performance, and identification of spot trains related to post-translational modifications.

Analysis of Variance↗
PubMed1997

Large-scale identification of proteins of Haemophilus influenzae by amino acid composition analysis.

Two-dimensional protein maps of microorganisms are useful tools for elucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. We applied amino acid composition analysis, following separation by two-dimensional gel electrophoresis, for large-scale identification of proteins of Haemophilus influenzae. H. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading frames, has been sequenced. For amino acid analysis, we used both precolumn derivatization of amino acids followed by reversed-phase chromatography of the derivatized residues and post-column derivatization of the residues previously separated on an ion exchanger. The composition analyses derived from both methods allowed the identification of 110 protein spots. The proteins were identified using the AACompldent software on the ExPASy server accessible via the World Wide Web with a success rate of 52%. In some cases, introduction of the analysis data of 12 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambiguously identified. Amino acid composition analysis proved to be an error-robust, efficient method for protein identification. The method can be practically established in every biochemical laboratory and, complementary to mass spectrometry, represents an important analytical tool for the mapping of the proteomes of organisms of interest.

Amino Acids↗
PubMed2003

Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation.

Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.

Albumins↗
PubMed2004

Target class strategies in mass spectrometry-based proteomics.

The cornerstone of proteomics resides in using traditional methods of protein chemistry, to extract and resolve complex mixtures, in concert with the powerful engines of mass spectrometry to decipher peptide and protein identities. The broad utility of proteomics technologies to map protein interactions, understand regulatory mechanisms and identify biomarkers associated with disease states and drug treatments necessitates a targeted biochemical approach tailored to the characteristics of the tissue, fluid or cellular extract being studied. The application of affinity methods in proteomic studies to focus on particular classes of molecules is being used with increasing frequency and comprises the subject of this review. An overview of successfully applied affinity methods is provided, along with speculation on the use of innovative approaches. Sample preparation and processing are critical for proteomics with affinity reagents, as only functional and active proteins can be isolated in most cases. Considerations for methods of sample preparation to optimize affinity capture and release are also discussed.

Animals↗
PubMed2005

Sociomics! Using the IssueCrawler to map, monitor and engage with the global proteomics research network.

We invite comment upon an experiment to locate proteomics on the WWW using a software tool called the IssueCrawler. We call our research "sociomics" because, like the bioscience omics, it is a semi-automated, computerised approach to the global analysis of data, whose computerised results can be integrated towards the development of a new "systems sociology" approach to the study of society. Our findings are that proteomics on the web is a scale-free network whose nodes display considerable "dynamic range".

Cluster Analysis↗
PubMed2002

Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolor.

The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their annotated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites.

Acetylation↗
PubMed2005

A human protein-protein interaction network: a resource for annotating the proteome.

Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

Axin Protein↗
PubMed1997

Of genomes and proteomes.

The era of complete genome sequences has arrived and with it vast amounts of data which must be annotated, cross referenced, and placed within the regulatory networks which define the physiology of an organism. One eucaryotic and three procaryotic genomes have been completed and the data made available and another 50 sequences are expected to be completed by the end of the decade. One of the first steps in the new post genome era will be to decipher the functions of the huge numbers of new open reading frames. Various approaches to investigate what unknown genes do and how genes interact together within an organism are being undertaken, including (1) the simultaneous measurement of the expression levels of all genes in a cell and (2) the mapping and quantitation of all proteins expressed within a cell. The idea of systematically mapping and identifying the total protein complement of the genome (the "proteome") arose over 20 years ago when the separation of proteins from total cell extracts by two dimensional (2D) gel electrophoresis was developed. This review will focus on the use of 2D gel electrophoresis as the basis for constructing proteome maps and on the rapid advances in mass spectrometry which will allow the large-scale, automated identification of proteins which is necessary for the creation of such databases.

Animals↗
PubMed2006

[Proteomic analysis of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells].

OBJECTIVE: To investigate the mechanism of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells. METHODS: In this study, the proteins extracted from paclitaxel-induced apoptotic MCF-7 cells were analyzed by 2-dimentional gel electrophoresis (2-DE), and compared with those from untreated MCF-7 cells. The differential proteins were identified by mass spectrometry. RESULTS: At 24 hour after paclitaxel (100 nmol/L) treatment, MCF-7 cells were collected and extracted the whole proteins. Seventeen up-regulated or down-regulated proteins were found by analysis of the differential proteomic 2-DE map. Six of them were identified by mass spectrometry. They were enolase 1, chloride intracellular channel 1, keratin 8, ribosomal protein S12, galectin-1 and histidine triad nucleotide binding protein, respectively. CONCLUSION: We effectively found the changed proteins in the process of paclitaxel-induced apoptosis in MCF-7 human breast carcinoma cells by proteomic techniques. These up-regulated or down-regulated proteins are important molecules for our further research about the mechanism of paclitaxel-induced apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Proteome analysis of cultured fibroblasts from type 1 diabetic patients and normal subjects.

CONTEXT: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. OBJECTIVE: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. DESIGN: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and alpha-actinin 4 and a decrease of tubulin-beta2 and splice isoform 2 of tropomyosin beta-chain were detected. CONCLUSIONS: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

Adult↗
PubMed2006

The Universal Protein Resource (UniProt): an expanding universe of protein information.

The Universal Protein Resource (UniProt) provides a central resource on protein sequences and functional annotation with three database components, each addressing a key need in protein bioinformatics. The UniProt Knowledgebase (UniProtKB), comprising the manually annotated UniProtKB/Swiss-Prot section and the automatically annotated UniProtKB/TrEMBL section, is the preeminent storehouse of protein annotation. The extensive cross-references, functional and feature annotations and literature-based evidence attribution enable scientists to analyse proteins and query across databases. The UniProt Reference Clusters (UniRef) speed similarity searches via sequence space compression by merging sequences that are 100% (UniRef100), 90% (UniRef90) or 50% (UniRef50) identical. Finally, the UniProt Archive (UniParc) stores all publicly available protein sequences, containing the history of sequence data with links to the source databases. UniProt databases continue to grow in size and in availability of information. Recent and upcoming changes to database contents, formats, controlled vocabularies and services are described. New download availability includes all major releases of UniProtKB, sequence collections by taxonomic division and complete proteomes. A bibliography mapping service has been added, and an ID mapping service will be available soon. UniProt databases can be accessed online at http://www.uniprot.org or downloaded at ftp://ftp.uniprot.org/pub/databases/.

Databases, Protein↗
PubMed2002

Mapping and structural dissection of human 20 S proteasome using proteomic approaches.

The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (alpha7beta7beta7alpha7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the alpha and beta subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the alpha7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser(250).

Amino Acid Sequence↗
PubMed2004

Protein interaction networks from yeast to human.

Protein interaction networks summarize large amounts of protein-protein interaction data, both from individual, small-scale experiments and from automated high-throughput screens. The past year has seen a flood of new experimental data, especially on metazoans, as well as an increasing number of analyses designed to reveal aspects of network topology, modularity and evolution. As only minimal progress has been made in mapping the human proteome using high-throughput screens, the transfer of interaction information within and across species has become increasingly important. With more and more heterogeneous raw data becoming available, proper data integration and quality control have become essential for reliable protein network reconstruction, and will be especially important for reconstructing the human protein interaction network.

Animals↗
PubMed2003

Normal human dermal fibroblasts: proteomic analysis of cell layer and culture medium.

Proteins present within the cell layer and those released in the cell medium from in vitro cultured normal human dermal fibroblasts were separated and characterized in terms of their isoelectric point and molecular weight, by two-dimensional (2-D) gel electrophoresis. All spots in the synthetic gel were firstly analyzed by the Melanie 3 software and compared with those of breast cancer cells, colorectal epithelial cells, HL60, lymphoma cells, and platelets, already available on-line. From the identification of 144 spots from both the cell layer and the medium, we were able to recognize 89 different proteins, since a certain number of spots represented different isoforms of the same molecule. Identifications were performed by matching with on-line 2-D databases, and by matrix assisted laser-desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), in order to confirm the identification by matching, or to identify new proteins. The procedure we used allows (i) to design a highly reproducible reference map of the proteome of adult human normal fibroblasts in culture, (ii) to evaluate protein species produced in the cell layer as well as those released in the culture medium, and (iii) to compare data from gel matching with those obtained by MS. This work represents an essential step for a better knowledge of mesenchymal cells, given the widespread use of this cell type in both clinical and experimental investigations.

Adult↗