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PubMed2001

Proteome analysis of the common human pathogen Helicobacter pylori.

The common human pathogen Helicobacter pylori is the major cause of gastric and duodenal ulcers. Based on the complete genome sequences of two independent isolates more than 1800 protein species have been resolved by two-dimensional gel electrophoresis and more than 200 of them have been identified (http://www.mpiib-berlin.mpg.de/2D-PAGE). Using these data, a large range of research areas including strain fingerprinting, protein composition and subcellular localization, gene regulation, and pathogen-host interactions have been investigated. The results that have been obtained led to a more detailed understanding of the Helicobacter biology and pathology and open further interesting fields for future work.

Bacterial Proteins↗
PubMed2005

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform.

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2002

A strategy to retrieve the whole set of protein modules in microbial proteomes.

Protein homology is often limited to long structural segments that we have previously called modules. We describe here a suite of programs used to catalog the whole set of modules present in microbial proteomes. First, the Darwin AllAll program detects homologous segments using thresholds for evolutionary distance and alignment length, and another program classifies these modules. After assembling these homologous modules in families, we further group families which are related by a chain of neighboring unrelated homologous modules. With the automatic analysis of these groups of families sharing homologous modules in independent multimodular proteins, one can split into their component parts many fused modules and/or deduce by logic more distant modules. All detected and inferred modules are reassembled in refined families. These two last steps are made by a unique program. Eventually, the soundness of the data obtained by this experimental approach is checked using independent tests. To illustrate this modular approach, we compared four proteobacterial proteomes (Campylobacter jejuni, Escherichia coli, Haemophilus influenzae, and Helicobacter pylori). It appears that this method might retrieve from present-day proteins many of the modules which can help to trace back ancient events of gene duplication and/or fusion.

Bacterial Proteins↗
PubMed2004

Functional proteomics mapping of a human signaling pathway.

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Proteomic analysis of the response of human lung cells to uranium.

The industrial use of uranium and particularly of depleted uranium, has pinpointed the need to review its chemical impact on human health. A proteomic approach was used to evaluate the response of a human lung cell line (A549) to uranium. We established the first 2-D reference map of the A549 cell line, identifying 87 spots corresponding to 81 major proteins. Uranium treatment triggered differential expression of 18 spots, of which 14 corresponded to fragments of cytokeratin 8 (CK8) and cytokeratin (CK18) and 1 to peroxiredoxin 1. We probed several hypotheses regarding CK cleavage, and observed that it did not result from caspase or calpain activity. Furthermore, we showed that the fragments are recognised by an anti-ubiquitin antibody (KM691). These results suggest a regulatory pathway involving CK ubiquitinylation or dysfunction in the proteasome-ubiquitin system in response to uranium exposure in human lung cells.

Amino Acid Sequence↗
PubMed2004

Cytoplasmic regulation of the accumulation of nuclear-encoded proteins in the mitochondrial proteome of maize.

Mitochondria from normal (NA)- and Texas (T)-cytoplasm maize (Zea mays L.) were purified from unpollinated ears via Percoll centrifugation. Approximately 300 mitochondrial proteins were resolved using two-dimensional (2-D) electrophoresis. The 197 most abundant proteins were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry involving overlapping pH gradients (pH 4-7 and 6-9). Database searches identified 58 genes that encode 100 of these protein spots. Functions could be predicted for 38 of the 58 genes (66%). All but one of these genes are located in the nuclear genome. Thirteen per cent of the analyzed protein spots (25 out of 197) exhibited at least a threefold difference in accumulation between the mitochondrial proteomes of NA- or T-cytoplasm maize plants that had essentially identical nuclear genomes. As most of these proteins were nuclear-encoded, these findings demonstrate that the genotype of a mitochondrion can regulate the accumulation of the nuclear-encoded fraction of its proteome. About half (27 out of 58) of the maize mitochondrial proteins identified in this study were not recovered in previous analyses of the Arabidopsis and rice mitochondrial proteomes.

Arabidopsis↗
PubMed2006

Proteomic analysis of rat liver phosphoproteins after treatment with protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide).

Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the current methods are limited by the use of antibodies, many of which do not cross-react between several species. The proteomics approach described herein has the potential to reveal novel tissue substrates, potential new pathway interconnections, and inhibitor specificity by monitoring differences in protein phosphorylation. We used the protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide) as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect treatment-related effects of a protein kinase A (PKA) inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput two-dimensional gel electrophoresis, differential gel staining with Pro-Q Diamond/SYPRO Ruby, statistical analysis, and matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis, we were able to show clear differences between the phosphoprotein profiles of rat liver protein extract from control and treated animals. Moreover, several proteins that show a potential change in phosphorylation were previously identified as PKA substrates or have putative PKA phosphorylation sites. The data presented support the use of differential proteomic methods to measure effects of kinase inhibitor treatment on protein phosphorylation in vivo.

Animals↗
PubMed2004

Two-dimensional protein database of human pancreas.

We report here the two-dimensional protein database of human pancreas. The proteins were analyzed by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Totally, 302 proteins were identified, of which about 27% were enzymes with a broad range of catalytic activities. Several of these are specifically expressed in pancreas, such as pancreatic amylase, pancreatic stone protein, pancreatitis-associated protein, pancreatic lipase, pancreatic elastase, etc. Structural and cytoskeletal proteins are also strongly represented on the gels. Thus, the pancreatic proteome reflects the organ's function. This work paves the way for further studies on pancreatic protein expression in health and disease, such as diabetes and pancreatic cancer.

Databases, Protein↗
PubMed2003

Proteomic characterization of metabolites, protein adducts, and biliary proteins in rats exposed to 1,1-dichloroethylene or diclofenac.

A proteome profiling approach was used to compare effects of two toxicants, 1,1-dicloroethylene (DCE) and diclofenac, which covalently adduct hepatic proteins. Bile was examined as a potential source of protein alterations since both toxicants target the hepatic biliary canaliculus. Bile was collected before and after toxicant treatment. Biliary proteins were separated by one-dimensional SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with data-dependent scanning. Comprehensive analysis of biliary proteins was performed by using SEQUEST and BLAST database searching, in combination with de novo interpretation. Bile not subjected to tryptic digestion was analyzed for DCE metabolites. DCE treatment resulted in a marked increase in the overall number of biliary proteins, whereas few changes in the proteomic profile were apparent in bile after diclofenac treatment. This is consonant with prior observations of more profound effects of DCE on canalicular membrane integrity. LC-MS-MS analyses for DCE metabolites revealed the presence of S-carboxymethyl glutathione, S-(cysteinylacetyl)glutathione, and a product of the intramolecular rearrangement of the DCE metabolite, ClCH(2)COSG, not previously described in vivo. In addition, several S-carboxymethylated proteins were identified in bile from DCE-treated animals. This investigation has produced the first comprehensive baseline characterization of the content of the rat biliary proteome and the first documentation of alterations in the proteome of bile by toxicant treatment. In addition, the results provide direct in vivo evidence for DCE metabolic routes proposed in the formation of covalent adducts.

Animals↗
PubMed2004

Global analysis of the cortical neuron proteome.

In this study, a multidimensional fractionation approach was combined with MS/MS to increase the capability of characterizing complex protein profiles of mammalian neuronal cells. Proteins extracted from primary cultures of cortical neurons were digested with trypsin followed by fractionation using strong cation exchange chromatography. Each of these fractions was analyzed by microcapillary reversed-phase LC-MS/MS. The analysis of the MS/MS data resulted in the identification of over 15,000 unique peptides from which 3,590 unique proteins were identified based on protein-specific peptide tags that are unique to a single protein in the searched database. In addition, 952 protein clusters were identified using cluster analysis of the proteins identified by the peptides not unique to a single protein. This identification revealed that a minimum of 4,542 proteins could be identified from this experiment, representing approximately 16% of all known mouse proteins. An evaluation of the number of false-positive identifications was undertaken by searching the entire MS/MS dataset against a database containing the sequences of over 12,000 proteins from archaea. This analysis allowed a systematic determination of the level of confidence in the identification of peptides as a function of SEQUEST cross correlation (Xcorr) and delta correlation (DeltaCn) scores. Correlation charts were also constructed to show the number of unique peptides identified for proteins from specific classes. The results show that low-abundance proteins involved in signal transduction and transcription are generally identified by fewer peptides than high-abundance proteins that play a role in maintaining mammalian cellular structure and motility. The results presented here provide the broadest proteome coverage for a mammalian cell to date and show that MS-based proteomics has the potential to provide high coverage of the proteins expressed within a cell.

Amino Acid Sequence↗
PubMed2003

Proteomic approach to understanding antibiotic action.

We have used proteomic technology to elucidate the complex cellular responses of Bacillus subtilis to antimicrobial compounds belonging to classical and emerging antibiotic classes. We established on two-dimensional gels a comprehensive database of cytoplasmic proteins with pIs covering a range of 4 to 7 that were synthesized during treatment with antibiotics or agents known to cause generalized cell damage. Although each antibiotic showed an individual protein expression profile, overlaps in the expression of marker proteins reflected similarities in molecular drug mechanisms, suggesting that novel compounds with unknown mechanisms of action may be classified. Indeed, one such substance, a structurally novel protein synthesis inhibitor (BAY 50-2369), could be classified as a peptidyltransferase inhibitor. These results suggest that this technique gives new insights into the bacterial response toward classical antibiotics and hints at modes of action of novel compounds. Such a method should prove useful in the process of antibiotic drug discovery.

Anti-Bacterial Agents↗
PubMed2005

The peptidomics concept.

Peptides are a paramount example of how nature diversifies from one single gene to release multiple, regulated functionalities at the desired sites and time. To achieve this, peptides are sequentially generated by a complex network of more than 500 proteases, acting at intracellular sites, upon secretion, in extracellular environments, and, finally, serving (regulated) degradation. This cycle of maturation, activation, and degradation points out that the peptidome is mechanistically linked to the proteome: the distribution between both is regulated by proteases and counter-regulated by protease inhibitors. Given the high diversity of peptides in living systems and their involvement in key regulatory processes, a need for improved peptide discovery, ideally combining peptide sequence identification with peptide profiling, has emerged. Standard proteomic approaches are not suitable for a systematic peptide analysis, since they do not cover the low molecular mass window. The new direction in proteomic research to analyse this "terra incognita" is peptidomics. This novel concept aims at the comprehensive visualization and analysis of small polypeptides, thus covering the mass range between proteomics and metabonomics. The pacemakers for the development of peptidomics technologies are modern mass spectrometry and bioinformatics. They are ideally suited for sensitive and comprehensive peptide analysis, especially in combination with the massive information content of today's genomic and transcriptomic databases. Given the high diversity of native peptides in living systems, clinical chemistry and modern medicine are the prime application areas. The discovery of relevant peptide biomarkers and drug targets will strongly benefit from peptidomics.

Animals↗
PubMed2003

Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers.

Quantitative or comparative proteome analysis was initially performed with 2-dimensional gel electrophoresis with the inherent disadvantages of being biased towards certain proteins and being labor intensive. Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, (13)C, (18)O, or (15)N, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification. While each of the different labeling methods has its advantages and disadvantages, the endoprotease (16)O-to-(18)O catalyzed oxygen exchange at the C-terminal carboxylic acid is extremely promising because of the specificity assured by the enzymatic reaction and the labeling of essentially every protease-derived peptide. We show here that this methodology is applicable to complex biological samples such as a subfraction of human plasma. Furthermore, despite the relatively small mass difference of 4 Da between the two labeled forms, corresponding to the exchange of two oxygen atoms by two (18)O isotopes, it is possible to quantify differentially labeled proteins on an ion trap mass spectrometer with a mass resolution of about 2000 in automated data dependent LC-MS/MS acquisition mode. Post column sample deposition on a MALDI target parallel to on-line ESI-MS/MS enables the analysis of the same compounds by means of ESI- and MALDI-MS/MS. This has the potential to increase the confidence in the quantification results as well as to increase the sequence coverage of potentially interesting proteins by complementary peptide ionization techniques. Additionally the paired y-ion signals in tandem mass spectra of (16)O/(18)O-labeled peptide pairs provide a means to confirm automatic protein identification results or even to assist de novo sequencing of yet unknown proteins.

Amino Acid Sequence↗
PubMed2004

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae.

Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.

Bacterial Proteins↗
PubMed2001

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Computational Biology↗
PubMed2005

SPD--a web-based secreted protein database.

With the improved secreted protein prediction approach and comprehensive data sources, including Swiss-Prot, TrEMBL, RefSeq, Ensembl and CBI-Gene, we have constructed secretomes of human, mouse and rat, with a total of 18 152 secreted proteins. All the entries are ranked according to the prediction confidence. They were further annotated via a proteome annotation pipeline that we developed. We also set up a secreted protein classification pipeline and classified our predicted secreted proteins into different functional categories. To make the dataset more convincing and comprehensive, nine reference datasets are also integrated, such as the secreted proteins from the Gene Ontology Annotation (GOA) system at the European Bioinformatics Institute, and the vertebrate secreted proteins from Swiss-Prot. All these entries were grouped via a TribeMCL based clustering pipeline. We have constructed a web-based secreted protein database, which has been publicly available at http://spd.cbi.pku.edu.cn. Users can browse the database via a GO assignment or chromosomal-location-based interface. Moreover, text query and sequence similarity search are also provided, and the sequence and annotation data can be downloaded freely from the SPD website.

Animals↗
PubMed2004

Proteomics-based sequence analysis of plant gene expression--the chloroplast transcription apparatus.

The chloroplast transcription apparatus has turned out to be more complex than anticipated, with core polypeptides surrounded by multiple accessory proteins of diverse, and in part unexpected, functions. At least two different RNA-binding proteins and several redox-responsive proteins are components of the major chloroplast RNA polymerase termed PEP-A. One of the key-regulatory factors has been identified as a Ser/Thr-specific protein kinase that is sensitive to SH group modification by glutathione and by this means is able to modulate transcription. The cloned plastid transcription kinase from mustard (Sinapis alba L.) has been assigned as a member of the (mostly nucleo-cytosolic) CK2 family and hence has been termed cpCK2. Despite its apparent role in mustard chloroplast transcription, until recently no data have been available for other plant species. Using the web database resources, we find evidence for an evolutionarily conserved role of this redox-sensitive plastid transcription factor.

Amino Acid Sequence↗
PubMed2005

Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations.

The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.

Gene Expression Profiling↗