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PubMed2006

Towards the proteome of Burkholderia cenocepacia H111: setting up a 2-DE reference map.

Polyphasic-taxonomic studies of the past decade have shown that the Burkholderia cepacia complex (Bcc) comprises at least nine species, which share a high degree of 16S rDNA (98-100%) sequence similarity but only moderate levels of DNA-DNA hybridization. Members of the Bcc are well known as opportunistic pathogens of plants, animals and humans but also as biocontrol and bioremediation agents. In this study intra-, surface-associated and extracellular proteins of B. cenocepacia H111, which was isolated from a cystic fibrosis patient, were examined by 2-DE coupled to MALDI-TOF MS. MS and MS/MS data were searched against a database comprising all currently available annotated proteins of genetically closely related strains. In total 642 proteins spots were successfully identified corresponding to 390 different protein species, which were classified into functional categories. The majority of these proteins could be linked to housekeeping functions in energy production, amino acid metabolism, protein folding, post-translational modification and turnover, and translation. Noteworthy is the fact that a significant number of truly secreted and membrane proteins were identified in the extracellular and surface-associated sub-proteomes. This indicates that the pre-fractionation protocol used in this study is a highly valuable strategy for unravelling the cellular location of the identified proteins.

Bacterial Proteins↗

PubMed2006

SELDI-TOF-MS of saliva: methodology and pre-treatment effects.

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.

Biomarkers↗

PubMed2003

Detecting protein sequence conservation via metric embeddings.

MOTIVATION: Comparing two protein databases is a fundamental task in biosequence annotation. Given two databases, one must find all pairs of proteins that align with high score under a biologically meaningful substitution score matrix, such as a BLOSUM matrix (Henikoff and Henikoff, 1992). Distance-based approaches to this problem map each peptide in the database to a point in a metric space, such that peptides aligning with higher scores are mapped to closer points. Many techniques exist to discover close pairs of points in a metric space efficiently, but the challenge in applying this work to proteomic comparison is to find a distance mapping that accurately encodes all the distinctions among residue pairs made by a proteomic score matrix. Buhler (2002) proposed one such mapping but found that it led to a relatively inefficient algorithm for protein-protein comparison. RESULTS: This work proposes a new distance mapping for peptides under the BLOSUM matrices that permits more efficient similarity search. We first propose a new distance function on peptides derived from a given score matrix. We then show how to map peptides to bit vectors such that the distance between any two peptides is closely approximated by the Hamming distance (i.e. number of mismatches) between their corresponding bit vectors. We combine these two results with the LSH-ALL-PAIRS-SIM algorithm of Buhler (2002) to produce an improved distance-based algorithm for proteomic comparison. An initial implementation of the improved algorithm exhibits sensitivity within 5% of that of the original LSH-ALL-PAIRS-SIM, while running up to eight times faster.

Algorithms↗

PubMed2005

Rice proteome database: a step toward functional analysis of the rice genome.

The technique of proteome analysis using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has the power to monitor global changes that occur in the protein complement of tissues and subcellular compartments. In this study, the proteins of rice were cataloged, a rice proteome database was constructed, and a functional characterization of some of the identified proteins was undertaken. Proteins extracted from various tissues and subcellular compartments in rice were separated by 2D-PAGE and an image analyzer was used to construct a display of the proteins. The Rice Proteome Database contains 23 reference maps based on 2D-PAGE of proteins from various rice tissues and subcellular compartments. These reference maps comprise 13129 identified proteins, and the amino acid sequences of 5092 proteins are entered in the database. Major proteins involved in growth or stress responses were identified using the proteome approach. Some of these proteins, including a beta-tubulin, calreticulin, and ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have unexpected functions. The information obtained from the Rice Proteome Database will aid in cloning the genes for and predicting the function of unknown proteins.

Calreticulin↗

PubMedDate not supplied

Toxicoproteomics: proteomics applied to toxicology and pathology.

Global measurement of proteins and their many attributes in tissues and biofluids defines the field of proteomics. Toxicoproteomics, as part of the larger field of toxicogenomics. seeks to identify critical proteins and pathways in biological systems that are affected by and respond to adverse chemical and environmental exposures using global protein expression technologies. Toxicoproteomics integrates 3 disciplinary areas: traditional toxicology and pathology, differential protein and gene expression analysis, and systems biology. Key topics to be reviewed are the evolution of proteomics, proteomic technology platforms and their capabilities with exemplary studies from biology and medicine, a review of over 50 recent studies applying proteomic analysis to toxicological research, and the recent development of databases designed to integrate -Omics technologies with toxicology and pathology. Proteomics is examined for its potential in discovery of new biomarkers and toxicity signatures, in mapping serum,plasma. and other biofluid proteomes, and in parallel proteomic and transcriptomic studies. The new field of toxicoproteomics is uniquely positioned toward an expanded understanding of protein expression during toxicity and environmental disease for the advancement of public health.

Animals↗

PubMed2002

Lens proteomics: analysis of rat crystallin sequences and two-dimensional electrophoresis map.

PURPOSE: To determine the sequence of four rat beta-crystallins, confirm the sequences by mass spectrometry, and produce a two-dimensional electrophoresis (2-DE) map of soluble crystallins in young rat lens. METHODS: New or additional sequences were determined for betaB1, betaB3, betaA3, and betaA4-crystallin cDNAs from Sprague-Dawley rats, and the deduced protein sequences confirmed by mass spectrometry. The identity and relative abundance of each crystallin was then determined by 2-DE of soluble protein from whole lenses of 12-day-old rats, image analysis, and tandem mass spectrometry (MS/MS) spectra of peptides from in-gel digests. RESULTS: The previously unreported sequence of rat betaA4 cDNA encoded a 195-amino-acid protein. Additional cDNA sequencing provided the previously unknown N-terminal sequence of rat betaA3, found two differences from the previous amino acid sequences of both rat betaB1 and betaB3, and detected a polymorphism at residue 54 in rat betaB3. These new sequences were then confirmed by whole protein masses and MS/MS spectra of proteolytic digests. 2-DE analysis provided a more detailed map of rat crystallins than previously available and allowed the composition of crystallins in young rat lens to be compared with that in young human lens. CONCLUSIONS: This report provides baseline data that will facilitate the analysis of posttranslational modifications in rat crystallins during cataract. Detection of a polymorphism in the sequence of rat betaB3 suggests that crystallins in humans could also exhibit polymorphisms. The unusual abundance of rat betaB3 and low abundance of betaB2 may account for the increased susceptibility of rat crystallins to insolubilization during aging and cataract.

Amino Acid Sequence↗

PubMed2006

Proteomic approach to substrates related to MAPK pathway in 293T cells.

Abundant evidence indicates that potential scaffold proteins and adaptor or linker molecules organize and specify various MAP kinase cascades. In the present study, proteomic methodologies were applied to screen these potential molecules in combination with cell morphology and cell cycle analysis. MEK1E, MKK3b, MKK5D and MKK7D were selected as representative MKKs of four main MAPK pathways. Our results showed that similar morphological transformation and G(2)/M cell cycle arrest were promoted by the over-expressed four kinases. Furthermore, global change in response to the over-expressed four kinases was characterized by differential proteomics. Eleven distinctly changed proteins were detected, in which four proteins (serine/threonine kinase 4, glutathione S-transferase p1-1, glycoprotein IX and soluble inorganic pyrophosphatase) were reported to be relative to MAPK pathways, while the other seven proteins may be new elements of substrates of the kinases. In our experiment, the expression of platelet glycoprotein IX precursor, glutathione S-transferase p1-1, peroxiredoxin 6, Ras-related protein Rab-34 and arginase II, mitochondrial precursor was up-regulated, while the expression of serine/threonine kinase 4 (MST1) was down-regulated by the four kinases. These results suggest that these six proteins may be common targets of all the MAPK pathways in 293T cell line. Interestingly, the expression of splicing factor 3B subunit 4 and soluble inorganic pyrophosphatase (Ppase) was specifically up-regulated by MEK1E and MKK5D, and by MEK1E, MKK3b and MKK5D, respectively. The expression of methylglyoxalase was down-regulated by MEK1E and MKK7D. Furthermore, the expression of ADP-ribosylation factor-like protein 1 was up-regulated by MKK5D but down-regulated by MKK3b and MKK7D. These findings revealed the characteristic molecular responses to four MKKs. In conclusion, our study not only confirms that MST1, glutathione S-transferase p1-1, glycoprotein IX and soluble PPase belong to MAPK pathways, but also provides seven novel molecules for the further study of the pathways.

Cell Cycle↗

PubMed2004

The complete genome and proteome of Mycoplasma mobile.

Although often considered "minimal" organisms, mycoplasmas show a wide range of diversity with respect to host environment, phenotypic traits, and pathogenicity. Here we report the complete genomic sequence and proteogenomic map for the piscine mycoplasma Mycoplasma mobile, noted for its robust gliding motility. For the first time, proteomic data are used in the primary annotation of a new genome, providing validation of expression for many of the predicted proteins. Several novel features were discovered including a long repeating unit of DNA of approximately 2435 bp present in five complete copies that are shown to code for nearly identical yet uniquely expressed proteins. M. mobile has among the lowest DNA GC contents (24.9%) and most reduced set of tRNAs of any organism yet reported (28). Numerous instances of tandem duplication as well as lateral gene transfer are evident in the genome. The multiple available complete genome sequences for other motile and immotile mycoplasmas enabled us to use comparative genomic and phylogenetic methods to suggest several candidate genes that might be involved in motility. The results of these analyses leave open the possibility that gliding motility might have arisen independently more than once in the mycoplasma lineage.

Amino Acid Sequence↗

PubMed2005

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.

Amino Acid Sequence↗

PubMed2004

Computational peptide dissection of Melan-a/MART-1 oncoprotein antigenicity.

We have mapped the linear antigenic determinant of a commercial MAb raised in the mouse against the melanoma-associated-antigen Melan-A/MART-1. The B cell epitope on the Melan-A/MART-1 oncoprotein is located in the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP, within residues 102-106. The definition of the antigenic sequence on Melan-A/MART-1 oncoprotein was reached following analyses of MHC II binding potential and similarity level to the mouse proteome, that put into evidence the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP as the top scoring peptide in binding H2-A(d) molecules and the epitopic sequence residues 102-106 (i.e., the peptide sequence PAYEK) as having low-similarity level to the mouse proteome. Dot-blot epitope mapping immunoassay identified proline residue 102 as critical, based on its effect on antibody recognition. The present study adds to previous companion reports in validating the hypothesis that low-similarity to the host's proteome and binding potential to MHC II molecules are essential concurring factors in the modulation of the pool of epitopic sequences.

Amino Acid Sequence↗

PubMed2006

Recent developments in proteomics: implications for the study of cardiac hypertrophy and failure.

The key components to the molecular understanding of the pathophysiology of various forms of heart failure involve global and/or large-scale identifications of proteins, their patterns of expression, posttranslational modifications, and functional characterization. Particularly, proteins involved in the induction of cardiac (mal)adaptive hypertrophic growth, interstitial fibrosis, and contractile dysfunction are of interest. In general, with the accumulation of vast amounts of DNA sequences in databases, researchers have become aware that merely having complete sequences of genomes and transcriptional changes for thousands of genes simultaneously will not be sufficient to elucidate, in molecular terms, the etiology and pathophysiology of cardiovascular disease. In the last decade, a new technology called proteomics has become available that allows biological and (patho)physiological questions to be approached exclusively from the protein perspective. Proteomics may enable us to map the entire complement of proteins expressed by the heart at any time and condition. This approach creates the unique possibility to identify, by differential analysis, protein alterations associated with the etiology of heart disease and its progression, outcome, and response to therapy. To illustrate the true power of proteomics, most of the currently available methodologies are first reviewed, including their limitations. This review also deals with the current status and the perspectives of proteomics applications in research on heart failure in general. Furthermore, examples of our recent data on global protein profiling of the pressure-overloaded rat right ventricle and of endothelin-1-stimulated cultures of neonatal rat cardiac myocytes are provided. The last section is devoted to the continuous advances in proteomic technologies, including protein separation methods, mass spectrometric instrumentation, computational analysis, and bioinformatic tools, together with integrative databases.

Cardiomegaly↗

PubMed2004

A Drosophila protein-interaction map centered on cell-cycle regulators.

BACKGROUND: Maps depicting binary interactions between proteins can be powerful starting points for understanding biological systems. A proven technology for generating such maps is high-throughput yeast two-hybrid screening. In the most extensive screen to date, a Gal4-based two-hybrid system was used recently to detect over 20,000 interactions among Drosophila proteins. Although these data are a valuable resource for insights into protein networks, they cover only a fraction of the expected number of interactions. RESULTS: To complement the Gal4-based interaction data, we used the same set of Drosophila open reading frames to construct arrays for a LexA-based two-hybrid system. We screened the arrays using a novel pooled mating approach, initially focusing on proteins related to cell-cycle regulators. We detected 1,814 reproducible interactions among 488 proteins. The map includes a large number of novel interactions with potential biological significance. Informative regions of the map could be highlighted by searching for paralogous interactions and by clustering proteins on the basis of their interaction profiles. Surprisingly, only 28 interactions were found in common between the LexA- and Gal4-based screens, even though they had similar rates of true positives. CONCLUSIONS: The substantial number of new interactions discovered here supports the conclusion that previous interaction mapping studies were far from complete and that many more interactions remain to be found. Our results indicate that different two-hybrid systems and screening approaches applied to the same proteome can generate more comprehensive datasets with more cross-validated interactions. The cell-cycle map provides a guide for further defining important regulatory networks in Drosophila and other organisms.

Animals↗

PubMed2006

Array based capillary IEF with a whole column image of laser-induced fluorescence in coupling to capillary RPLC as a comprehensive 2-D separation system for proteome analysis.

Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.

Animals↗

PubMed2006

Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach.

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3-10 and 4-7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7', the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.

Animals↗

PubMed2002

Structural proteomics: developments in structure-to-function predictions.

The major challenge for post-genomic research is to functionally assign and validate a large number of novel target genes and their corresponding proteins. Functional genomics approaches have, therefore, gained considerable attention in the quest to convert this massive data set into useful information. One of the crucial components for the functional understanding of unassigned proteins is the analysis of their experimental or modeled 3D structures. Structural proteomics initiatives are generating protein structures at an unprecedented rate but our current knowledge of 3D-structural space is still limited. Estimates on the completeness of the 3D-structural coverage of proteins vary but it is generally accepted that only a minority of the structural proteome has a template structure from which reliable conclusions can be drawn. Thus, structural proteomics has set out to build a map of protein structures that will represent all protein folds included in the 'global proteome'.

Amino Acid Sequence↗

PubMed2000

PPMdb: a plant plasma membrane database.

PPMdb is a proteome database dedicated to proteins from plant plasma membranes. It provides comprehensive two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) maps, partial amino acid sequences and expression data. All this information is gathered and structured in a relational database, after being analyzed and annotated. PPMdb includes active links to related biological databases (EMBL, GenBank, GenPep, and SWISS-PROT and TrEMBL) as well as to MEDLINE abstracts. Information on specific protein spots can be displayed by clicking on the 2-D maps. In addition, users can query the database by accession number, protein name, pI and MW, and cellular location. Access to PPMdb is available at the following URL: http://sphinx.rug. ac.be:8080.

Amino Acid Sequence↗

PubMed2004

Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips.

The TOR (target of rapamycin) proteins play important roles in nutrient signaling in eukaryotic cells. Rapamycin treatment induces a state reminiscent of the nutrient starvation response, often resulting in growth inhibition. Using a chemical genetic modifier screen, we identified two classes of small molecules, small-molecule inhibitors of rapamycin (SMIRs) and small-molecule enhancers of rapamycin (SMERs), that suppress and augment, respectively, rapamycin's effect in the yeast Saccharomyces cerevisiae. Probing proteome chips with biotinylated SMIRs revealed putative intracellular target proteins, including Tep1p, a homolog of the mammalian PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function that we show to be a component of the TOR signaling network. Both SMIR target proteins are associated with PI(3,4)P2, suggesting a mechanism of regulation of the TOR pathway involving phosphatidylinositides. Our results illustrate the combined use of chemical genetics and proteomics in biological discovery and map a path for creating useful therapeutics for treating human diseases involving the TOR pathway, such as diabetes and cancer.

Humans↗

PubMed2005

[Two-dimensional liquid chromatographic fractionation of mouse liver phosphoproteome].

OBJECTIVE: To fractionate mouse liver phosphoproteome by two-dimensional liquid chromatography. METHODS: The phosphoproteins were extracted from the lysates of normal mouse livers with phosphate metal affinity chromatography resin. The phosphoproteins were exchanged by the initial buffer and separated by chromatofocusing in the first dimension. The fractions with pH value between 8.5 and 4.0 were separated by non-porous silica reverse-phase high-performance liquid chromatography. Finally, the UV maps were converted into gel maps by ProteoVue software. RESULTS: The phosphoproteins of mouse liver were successfully extracted and fractionated by two-dimensional liquid chromatographic fractionation after concentration and desalting. The pI/UV map of mouse liver phospho-proteomic was obtained successfully. There were 16 fractions with pH between 8.5 and 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel maps. CONCLUSION: Combination of phosphoprotein enrichment and two-dimensional liquid chromatographic fractionation is an effective approach of phosphoproteomic studies.

Animals↗