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PubMed2005

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2.

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.

Amino Acid Sequence↗

PubMed2005

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome.

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.

Amino Acid Sequence↗

PubMed2006

Proteome profile changes that are differentially regulated by lipid and protein phosphatase activities of tumor suppressor PTEN in PTEN-expressing U-87 MG human glioblastoma cells.

The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.

Blotting, Western↗

PubMed2006

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032.

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.

Amino Acid Sequence↗

PubMed2006

Proteome analysis of human lung squamous carcinoma.

Few lung cancer-specific molecular markers have been established in regard of "early-stage" diagnosis and prognosis. In this study the proteome analysis of human lung squamous carcinoma (hLSC) was carried out using two strategies to explore the carcinogenic mechanisms and identify its molecular markers more directly and comprehensively. Comparative proteome analysis on 20 hLSC tissues and paired normal bronchial epithelial tissues revealed 76 differential proteins, among which 68 proteins were identified by PMF. The identified proteins fell into three categories: oncoproteins, cell cycle regulators and signaling molecules. To validate the identified differential proteins, the expressions levels of three differential proteins mdm2, c-jun and EGFR were determined by immunohistochemical staining and immunoblots. The results verified proteome analysis results. Serological proteome analysis (SERPA) of ten hLSC tissues was performed to identify the tumor-associated antigens. The results revealed 36 +/- 8 differential proteins reactive with patients' autologous sera, of which 14 proteins were identified. Six of the 14 proteins, alpha enolase, pre-B cell-enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose-bisphosphate aldolase A, and guanine nucleotide-binding protein beta subunit-like protein, were also up-regulated in hLSCs in the comparative proteomic study, which suggests potential application of these 6 hLSC-associated antigens in diagnosis and therapy of hLSC.

Aged↗

PubMed2005

Report on the first congress of the Spanish Proteomics Society.

This report describes the first congress of the Spanish Proteomics Society (SEProt) and the Foundation Meeting of the European Proteomics Association (EuPA), which were held in Cordoba, Spain, February 13-17, 2005. The EuPA meeting joined together thirty European representatives from 17 national European proteomics organisations to discuss the opportunity of creating a supranational association aimed to promote all aspects of proteomics as a scientific discipline throughout Europe. The main subjects of the SEProt congress, which was attended by over 350 researchers mainly from Spain and other European countries, were the current status and applications of proteomics in medical and biological sciences.

Congresses as Topic↗

PubMed2006

Identification of a plasma proteomic signature to distinguish pediatric osteosarcoma from benign osteochondroma.

Osteosarcoma (OS) is the most common malignant bone tumor in children. To identify a plasma proteomic signature that can detect OS, we used SELDI MS to perform proteomic profiling on plasma specimens from 29 OS and 20 age-matched osteochondroma (OC) patients. Nineteen statistically significant ion peaks that were differentially expressed in OS when compared with OC patients were identified (p < 0.001 and false discovery rate < 10%). Using the proteomic profiles, we constructed a multivariate 3-nearest neighbors classifier to distinguish OS from OC patients with a sensitivity of 97% and a specificity of 80% based on external leave-one-out crossvalidation. Permutation test showed that the classification result was statistically significant (p < 0.00005). One of the proteins (m/z 11 704) in the proteomic signature was identified as serum amyloid protein A (SAA) by PMF. The higher plasma level of SAA in OS patients was further validated by Western blotting when compared to that of osteochrondroma patients and normal subjects as reference. The classifier based on this plasma proteomic signature may be useful to differentiate malignant bone cancer from benign bone tumors and for early detection of OS in high-risk individuals.

Adolescent↗

PubMed2006

A proteomic approach for dissecting H-Ras signaling networks in NIH/3T3 mouse embryonic fibroblast cells.

To elucidate an understanding into H-Ras protein network, we have established various oncogene H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which are expressing G12V H-Ras, G12R H-Ras, and G12V/T35S H-Ras proteins under the tight control of expression by an antibiotic doxycycline. Here we provide a catalog of proteome profiles in total cell lysate derived from the oncogenic and partial loss of function H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF-MS analysis both commonly in oncogenic and partial loss of function H-Ras expression system. Thus, we tried to dissect H-Ras signaling pathway, especially a downstream effector molecule, Raf in NIH/3T3 cells using proteomics tools. In this study, we centralized upon the proteome profile changes as common targets for oncogenic H-Ras and a partial loss of function H-Ras in the H-Ras-expressing cells. Thirteen protein spots were selected as what the staining intensities on the gels for 2-DE images from both kinds of cells were consistently changed in their protein expression level. Differentially regulated expression was further confirmed for some subsets of candidates by semiquantitative RT-PCR and Western blot analysis using specific antibodies. Taken together, our results obtained and present here show that the comparative analysis of proteome from oncogenic and partial loss of function H-Ras-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly.

Animals↗

PubMed2006

The HUPO Plasma Proteome Project: a report from the Munich congress.

The Human Proteome Organization has several major collaborative research initiatives, including the Plasma Proteome Project. A major feature of the HUPO World Congress in Munich in August 2005 was the release of the special issue of PROTEOMICS with 28 articles from the pilot phase of the Plasma Proteome Project. An open Workshop and a presentation in the closing plenary session of the congress focused on next phases for the Plasma Proteome Project.

Antibodies↗

PubMed2006

The proteome of Mannheimia succiniciproducens, a capnophilic rumen bacterium.

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is an industrially important bacterium as an efficient succinic acid producer. Recently, its full genome sequence was determined. In the present study, we analyzed the M. succiniciproducens proteome based on the genome information using 2-DE and MS. We established proteome reference map of M. succiniciproducens by analyzing whole cellular proteins, membrane proteins, and secreted proteins. More than 200 proteins were identified and characterized by MS/MS supported by various bioinformatic tools. The presence of proteins previously annotated as hypothetical proteins or proteins having putative functions were also confirmed. Based on the proteome reference map, cells in the different growth phases were analyzed at the proteome level. Comparative proteome profiling revealed valuable information to understand physiological changes during growth, and subsequently suggested target genes to be manipulated for the strain improvement.

Animals↗

PubMed2006

Guidelines for the next 10 years of proteomics.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

Biomarkers↗

PubMed2006

Protein Equalizer Technology : the quest for a "democratic proteome".

No proteome can be considered "democratic", but rather "oligarchic", since a few proteins dominate the landscape and often obliterate the signal of the rare ones. This is the reason why most scientists lament that, in proteome analysis, the same set of abundant proteins is seen again and again. A host of pre-fractionation techniques have been described, but all of them, one way or another, are besieged by problems, in that they are based on a "depletion principle", i.e. getting rid of the unwanted species. Yet "democracy" calls not for killing the enemy, but for giving "equal rights" to all people. One way to achieve that would be the use of "Protein Equalizer Technology" for reducing protein concentration differences. This comprises a diverse library of combinatorial ligands coupled to spherical porous beads. When these beads come into contact with complex proteomes (e.g. human urine and serum, egg white, and any cell lysate, for that matter) of widely differing protein composition and relative abundances, they are able to "equalize" the protein population, by sharply reducing the concentration of the most abundant components, while simultaneously enhancing the concentration of the most dilute species. It is felt that this novel method could offer a strong step forward in bringing the "unseen proteome" (due to either low abundance and/or presence of interference) within the detection capabilities of current proteomics detection methods. Examples are given of equalization of human urine and serum samples, resulting in the discovery of a host of proteins never reported before. Additionally, these beads can be used to remove host cell proteins from purified recombinant proteins or protein purified from natural sources that are intended for human consumption. These proteins typically reach purities of the order of 98%: higher purities often become prohibitively expensive. Yet, if incubated with "equalizer beads", these last impurities can be effectively removed at a small cost and with minute losses of the main, valuable product.

Biomarkers↗

PubMed2006

Advances in plant proteomics.

With the avalanche of genomic information and improvements in analytical technology, proteomics is becoming increasingly important for the study of many different aspects of plant functions. Since proteins serve as important components of major signaling and biochemical pathways, studies at protein levels are essential to reveal molecular mechanisms underlying plant growth, development, and interactions with the environment. The plant proteome is highly complex and dynamic. Although great strides need to be taken towards the ultimate goal of characterizing all the proteins in a proteome, current technologies have provided immense opportunities for high-throughput proteomic studies that have gone beyond simple protein identification to analyzing various functional aspects, such as quantification, PTM, subcellular localization, and protein-protein interactions. In this review of plant proteomics, advances in protein fractionation, separation, and MS will be outlined. Focus will be on recent development in functional analysis of plant proteins, which paves the way towards the comprehensive integration with transcriptomics, metabolomics, and other large scale "-omics" into systems biology.

Algorithms↗

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↗

PubMed2006

Proteome analysis of bronchoalveolar lavage in lung diseases.

The proteomic approach is complementary to genomics and enables protein composition to be investigated under various clinical conditions. Its application to the study of bronchoalveolar lavage (BAL) is extremely promising. BAL proteomic studies were initially based on two-dimensional electrophoretic separation of complex protein samples and subsequent identification of proteins by different methods. With the techniques available today it is possible to attain many different research objectives. BAL proteomics can contribute to the identification of proteins in alveolar spaces with possible insights into pathogenesis and clinical application for diagnosis, prognosis and therapy. Many proteins with different functions have already been identified in BAL. Some could be biomarkers that need to be individually confirmed by correlation with clinical parameters and validation by other methods on larger cohorts of patients. The standardization of BAL sample preparation and processing for proteomic studies is an important goal that would promote and facilitate clinical applications. Here, we review the principal literature on BAL proteomic analysis applied to the study of lung diseases.

Bronchoalveolar Lavage Fluid↗

PubMed2006

Geographical focus. Proteomics initiatives in Spain: ProteoRed.

The Spanish National Network of Proteomic Facilities--ProteoRed has been created as an initiative for the coordination, integration and development of the proteomics facilities and laboratories distributed throughout Spain. ProteoRed's main objective is to give support to the scientific community allowing them wide access to emerging proteomics technologies and thus encouraging the science of proteomics. In addition, standardization of protocols and robustness of workflows are addressed by multi-centric laboratory activities. Educational, training and dissemination issues are part of the core activities of ProteoRed. To reach these objectives, specific activities have been developed through six working groups (WG1-WG6) covering functional, technical, educational and scientific aspects of proteomics.

Computational Biology↗

PubMed2004

Proteomic analysis of mouse liver for the evaluation of effects of Scutellariae radix by liquid chromatography with tandem mass spectrometry.

Scutellariae radix or Scutellaria baicalensis is a medicinal plant that contains major flavonoids such as baicalein, baicalin, wogonin and wogonosides. The present work describes the development of an approach using proteomic analysis of mouse liver to study the effects of prolonged exposure to substances present in chemically standardized Scutellariae radix extracts. Histopathological examination of the mouse liver was compared with the proteome data. The botanical extracts were prepared using pressurized liquid extraction (PLE). A method without isotope labeling was developed, using proteolytic digestion with one- and two-dimensional liquid chromatography with tandem mass spectrometry, and was used to characterize the extent of differential protein expression in mouse liver in response to external factors such as extracts from Scutellariae radix. From the histopathological examination and proteome data, significant changes in the mouse livers were not observed for the low-dose group. The Scutellariae radix extracts at high dose were observed to cause damage at the bile duct and expression change of a number of proteins including some involved in catabolism of triglyceride-rich particles, carbohydrate metabolism, regulators of cell signaling processes, and enzymes involved in biotransformation. Thus, proteomic analysis of liver samples from mice treated with botanical extracts is a promising approach to provide information on any potential toxicity effects of the extracts. The present method also provides another means for comparing proteomes in biological samples such as liver lysates from mice subjected to different treatments.

Animals↗

PubMed2025

Proximity Proteomics to Profile Ebola Virus Protein Interactome in Its Functional Context.

Proximity labeling-based proteomics (proximity proteomics) has emerged as a popular and versatile approach to illuminate the molecular interactions between viruses and their hosts. In this approach, a proximity labeling enzyme tag is fused to a bait protein and labels neighboring proteins with a chemical handle such as biotin, allowing for downstream affinity purification. Compared to another widely used technique, affinity purification coupled mass spectrometry, proximity proteomics enables the detection of low affinity or transient interactors that might have important functions in the viral life cycle. Further, proximity proteomics can identify interactors of a labile bait protein, of which affinity purification is technically challenging. Here, we describe a proximity proteomic protocol to identify cellular interactors of the Ebola virus polymerase. A similar strategy is readily applicable to elucidate the virus-host interactions for Marburg virus.

Ebolavirus↗