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At least 523 records · Page 29Linked to original sources
PubMed2006

Immunoproteome analysis of soluble and membrane proteins of Shigella flexneri 2457T.

AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.

Animals↗
PubMed2001

Is there a bias in proteome research?

Advances in technology have enabled us to take a fresh look at data acquired by traditional single experiments and to compare them with genomewide data. The differences can be tremendous, as we show here, in the field of proteomics. We have compared data sets of protein-protein interactions in Saccharomyces cerevisiae that were detected by an identical underlying technical method, the yeast two-hybrid system. We found that the individually identified protein-protein interactions are considerably different from those identified by two genomewide scans. Interacting proteins in the pooled database from single publications are much more closely related to each other with respect to transcription profiles when compared to genomewide data. This difference may have been introduced by two factors: by a selection process in individual publications and by false positives in the whole-genome scans. If we assume that the differences are a result of false positives in the whole-genome data, the scans would contain 47%, 44%, and 91% of false positives for the UETZ, ITO-core, and ITO-full data, respectively. If, however, the true fraction of false positives is considerably lower than estimated here, the data from hypothesis-driven experiments must have been subjected to a serious selection process.

False Positive Reactions↗
PubMed2006

Proteomic profiling of fibroblasts reveals a modulating effect of extracellular calumenin on the organization of the actin cytoskeleton.

CREC proteins constitute a family of EF-hand calcium binding proteins localized to the secretory pathway. Calumenin is the only member known to be secreted. Recently, it was shown that thrombin-activated thrombocytes liberate calumenin, which also is found in atherosclerotic lesions but not in normal vasculature. To study the possible effects of calumenin extracellularly, we used proteomic profiling of fibroblasts cultured in absence and in presence of calumenin. Using 2-DE and MS/MS, we show that normal fibroblasts contain several 28-29-kDa N-terminal and a 16-kDa C-terminal fragment of beta- or gamma-actin. Extracellularly added calumenin decreases the levels of both the N-terminal and C-terminal actin fragments, and, in addition, decreases the expression level of septin 2, which interacts with the actin cytoskeleton and is involved in cytokinesis. Labeling of S-phase fibroblasts with bromo-2'deoxy-uridine indicates that calumenin added to the medium also modulates the cell cycle. Our study thus indicates that calumenin may have an autocrine or a paracrine effect on the cells in its vicinity, and, therefore, may be involved in the pathophysiology of thrombosis or in wound healing.

Actins↗
PubMed2004

Profiling the expression of mitogen-induced T-cell proteins by using multi-membrane dot-blotting.

High throughput technologies are standard methods for analysis of the proteome. Multi-layer multi-well plate dot-blotting system (MLDot) technology is a high-throughput dot blotting system that provides a simple, cost-effective approach for protein expression profiling in multiple samples. In contrast to traditional dot blot, MLDot uses a layered stack of thin, sieve-like membranes in place of a single nitrocellulose membrane. Therefore, up to 10 membranes can be prepared from the samples arrayed in a single 96-well plate. We describe the ability of MLDot to detect the predicted changes in protein expression following multiple mitogen treatment of T-cells. We compare the levels of the phopshorylated forms of CREB, Jun, and Akt in Jurkat T-cells as detected by MLDot to those measured by a gel-based assay. We also describe the ability of MLDot to detect differences in the levels of phosphorylated Akt in Jurkat cells as compared to primary lymphocytes.

Biotinylation↗
PubMed2001

Construction of a Francisella tularensis two-dimensional electrophoresis protein database.

We have started the construction of a two-dimensional database of the proteome of Francisella tularensis, a bacterium that is responsible for the highly pathogenic disease tularemia. The genome of this intracellular pathogen is not completely sequenced yet and, currently, information about only 66 proteins is available from NCBI database. We have analyzed the F. tularensis live vaccine strain by two-dimensional gel electrophoresis with immobilized pH 3-10 gradient in the first dimension and 9-16% gradient or tricine SDS-PAGE in the second dimension. In both cases about 2000 spots were detected. Furthermore, we compared the protein pattern of the nonvirulent F. tularensis live vaccine strain with protein profiles of two wild type clinical isolates and more than 50 differentially expressed proteins were counted. The separated proteins are going to be identified by peptide mass fingerprinting. However, due to the lack of complete genome sequence data only eight proteins were unambiguously identified. Among them, acid phosphatase and the most basic isoform of a hypothetical 23 kDa protein are characteristic only for virulent strains.

Bacterial Proteins↗
PubMed2005

Quantitative analysis of cytochrome p450 isozymes by means of unique isozyme-specific tryptic peptides: a proteomic approach.

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.

Animals↗
PubMed2006

Protein expression of lymphocytes in HLA-DR transgenic pigs by a proteomic approach.

Matching donor and recipient human leucocyte antigen (HLA-II) could conquer cell-mediated rejection following transplantation. Transgenic pigs carrying HLA genes that "humanize" porcine organs, tissues, and cells were successfully generated. This study further clarifies the effect of HLA-DR transgenes on lymphocyte protein expression, via a proteomic approach. Lymphocytes were isolated from two HLA-DR transgenic pigs and three nontransgenic littermates on 157 d after birth. Soluble protein of 1x10(7) cells was separated using 2-DE. In total, 301 colloidal CBB-stained protein spots detected on all five 2-D gels were quantified. Thirty-three proteins were differentially expressed by a factor of 1.5. These proteins were subsequently identified by MALDI-TOF MS and MALDI-TOF/TOF MS/MS. These proteins were sorted into the following categories: chaperones, T-lymphocyte function, DNA/RNA processing, cytoskeleton-associated proteins, signal transduction, enzymes, and unknown. Previous studies have suggested that some of the identified proteins are associated with lymphocyte activation/proliferation. The identities of the unidentified spots and the systematic effect of these up- and down-regulated proteins on T-cell function in HLA-DR transgenic pigs require further exploration.

Animals↗
PubMed2006

Protein-protein interactions among Helicobacter pylori cag proteins.

Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.

Adenosine Triphosphatases↗
PubMed2004

CEBS object model for systems biology data, SysBio-OM.

MOTIVATION: To promote a systems biology approach to understanding the biological effects of environmental stressors, the Chemical Effects in Biological Systems (CEBS) knowledge base is being developed to house data from multiple complex data streams in a systems friendly manner that will accommodate extensive querying from users. Unified data representation via a single object model will greatly aid in integrating data storage and management, and facilitate reuse of software to analyze and display data resulting from diverse differential expression or differential profile technologies. Data streams include, but are not limited to, gene expression analysis (transcriptomics), protein expression and protein-protein interaction analysis (proteomics) and changes in low molecular weight metabolite levels (metabolomics). RESULTS: To enable the integration of microarray gene expression, proteomics and metabolomics data in the CEBS system, we designed an object model, Systems Biology Object Model (SysBio-OM). The model is comprehensive and leverages other open source efforts, namely the MicroArray Gene Expression Object Model (MAGE-OM) and the Proteomics Experiment Data Repository (PEDRo) object model. SysBio-OM is designed by extending MAGE-OM to represent protein expression data elements (including those from PEDRo), protein-protein interaction and metabolomics data. SysBio-OM promotes the standardization of data representation and data quality by facilitating the capture of the minimum annotation required for an experiment. Such standardization refines the accuracy of data mining and interpretation. The open source SysBio-OM model, which can be implemented on varied computing platforms is presented here. AVAILABILITY: A universal modeling language depiction of the entire SysBio-OM is available at http://cebs.niehs.nih.gov/SysBioOM/. The Rational Rose object model package is distributed under an open source license that permits unrestricted academic and commercial use and is available at http://cebs.niehs.nih.gov/cebsdownloads. The database and interface are being built to implement the model and will be available for public use at http://cebs.niehs.nih.gov.

Database Management Systems↗
PubMed2007

Identification of proteomic changes during differentiation of adult mouse subventricular zone progenitor cells.

The use of neural precursor cells (NPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features that control NPC proliferation and their differentiation into neurons, astrocytes, and oligodendrocytes are unclear. In the present study, we used a comparative proteomics approach to identify proteins that were differentially regulated in NPCs after short-term differentiation. We also used a subcellular fractionation technique for enrichment of nuclei and other dense organelles to identify proteins that were not readily detected in whole cell extracts. In total, 115 distinct proteins underwent expression changes during NPC differentiation. Forty one of these were only identified following subcellular fractionation. These included transcription factors, RNA-processing factors, cell cycle proteins, and proteins that translocate between the nucleus and cytoplasm. Biological network analysis showed that the differentiation of NPCs was associated with significant changes in cell cycle and protein synthesis machinery. Further characterization of these proteins could provide greater insight into the mechanisms involved in regulation of neurogenesis in the adult central nervous system (CNS) and potentially identify points of therapeutic intervention.

Adult Stem Cells↗
PubMed2004

A statistical framework for combining and interpreting proteomic datasets.

MOTIVATION: To identify accurately protein function on a proteome-wide scale requires integrating data within and between high-throughput experiments. High-throughput proteomic datasets often have high rates of errors and thus yield incomplete and contradictory information. In this study, we develop a simple statistical framework using Bayes' law to interpret such data and combine information from different high-throughput experiments. In order to illustrate our approach we apply it to two protein complex purification datasets. RESULTS: Our approach shows how to use high-throughput data to calculate accurately the probability that two proteins are part of the same complex. Importantly, our approach does not need a reference set of verified protein interactions to determine false positive and false negative error rates of protein association. We also demonstrate how to combine information from two separate protein purification datasets into a combined dataset that has greater coverage and accuracy than either dataset alone. In addition, we also provide a technique for estimating the total number of proteins which can be detected using a particular experimental technique. AVAILABILITY: A suite of simple programs to accomplish some of the above tasks is available at www.unm.edu/~compbio/software/DatasetAssess

Algorithms↗
PubMed2006

Considerations for proteolytic labeling-optimization of 18O incorporation and prohibition of back-exchange.

Proteolytic (18)O labeling is a very powerful tool for differential analysis applied to proteome studies. However, it is a relatively new technique and the optimization of the labeling process still needs some attention. We found that the two-step post-proteolytic labeling should be favored over the conventional digestion of proteins in H(2) (18)O, since the former allows for higher sample concentrations and thus more favorable kinetics. It was demonstrated that the inhibitory effect of urea on (18)O incorporation could be compensated by the use of higher sample concentrations. Furthermore, it was shown that heat-deactivation of trypsin prevents (18)O/(16)O back-exchange. In addition, no non-specific hydrolysis of the peptides could be observed as a result of the heating. Heat inactivation of trypsin opens the way for the use of capillary electrophoresis as a separation technique in proteolytic labeling studies, as it abolishes the need for use of detrimental additives. Analysis of a labeled protein digest by capillary isoelectric focusing/mass spectrometry showed the applicability of the method. No back-exchange was observed across the entire electropherogram.

Electrophoresis, Capillary↗
PubMed2006

Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin.

Thioredoxin (TRX) is a small multifunctional protein with a disulfide active site involved in redox regulation. To gain insight into the numerous proteins able to interact with thioredoxin in Arabidopsis thaliana, we have compared three different proteomic procedures. In the two first approaches targets present in a mixture of soluble leaf proteins were reduced by the cytosolic TRX h3, then the new thiols were labeled either with radioactive iodoacetamide allowing specific detection (first method) or with a biotinylated thiol-specific compound allowing selective retention on an avidin column (second method). The third method involved a chromatography on a mutated TRX h3 column, which is able to covalently trap potential targets. All together, the three approaches enabled us to propose 73 proteins as being TRX-linked, and involved in various processes. Methods 1 and 3 were not only efficient with respectively 47 and 41 potential targets, but also complementary as only 26% of the targets were identified by both procedures. The second method with only 12 proteins was less efficient. However, this approach, as well as the first one when coupled with differential labeling of the cysteine residues, could be more informative about the cysteines involved in the thiol-disulfide interchange.

Arabidopsis↗
PubMed2005

Modulation of cytochrome C oxidase-va is possibly involved in metallothionein protection from doxorubicin cardiotoxicity.

Previous studies using a cardiac-specific metallothionein (MT)-overexpressing transgenic (MT-TG) mouse model have demonstrated that MT protects from doxorubicin (DOX)-induced oxidative heart injury. The molecular mechanisms that underlie this cardioprotection, however, have yet to be defined. In the present study, we tested the hypothesis that MT overexpression activates cytoprotective mechanisms, leading to cardiac protection from DOX toxicity. MT-TG mice and nontransgenic wild-type (WT) controls were treated i.p. with DOX at a single dose of 20 mg/kg and sacrificed on the third day after the treatment. An expression proteomic analysis involving two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify MT-induced changes in cytoprotection-related proteins. We identified 18 proteins that were modified by DOX treatment in the heart. These proteins included those involved in cellular antioxidant defense, enzymes of the mitochondrial electron transport chain, enzymes involved in beta-oxidation of fatty acids and glycolysis, and proteins involved in regulation of cardiac muscle contraction. However, the most dominant modification by MT is the cytochrome c oxidase subunit Va (CCO-Va). In response to DOX treatment, a specific isoform of CCO-Va was enhanced in the MT-TG but not in the WT mouse hearts. Because CCO-Va is a critical component in the mitochondrial electron transport chain, the results suggest that the cardioprotective effect of MT may be related to an increased expression or a differential modification of CCO-Va.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of slow- and fast-twitch skeletal muscles.

Skeletal muscles are composed of slow- and fast-twitch muscle fibers, which have high potential in aerobic and anaerobic ATP production, respectively. To investigate the molecular basis of the difference in their functions, we examined protein profiles of skeletal muscles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis with pH 4-7 and 6-11 isoelectric focusing gels. A comparison between rat soleus and extensol digitorum longus (EDL) muscles that are predominantly slow- and fast-twitch fibers, respectively, showed that the EDL muscle had higher levels of glycogen phosphorylase, most glycolytic enzymes, glycerol 3-phosphate dehydrogenase, and creatine kinase; while the soleus muscle had higher levels of myoglobin, TCA cycle enzymes, electron transfer flavoprotein, and carbonic anhydrase III. The two muscles also expressed different isoforms of contractile proteins including myosin heavy and light chains. These protein patterns were further compared with those of red and white gastrochnemius as well as red and white quadriceps muscles. It was found that metabolic enzymes showed a concerted regulation dependent on muscle fiber types. On the other hand, expression of contractile proteins seemed to be independent of the metabolic characteristics of muscle fibers. These results suggest that metabolic enzymes and contractile proteins show different expression patterns in skeletal muscles.

Animals↗
PubMed2006

Proteomic identification of glucocorticoid receptor interacting proteins.

The glucocorticoid receptor (GR) acts as a ligand dependent transcription factor but can also cross talk with other signaling pathways via protein-protein interactions. In this paper we describe methods to study novel cytosolic GR interacting proteins, using mAb based immunoaffinity chromatography of GR from rat liver cytosol. Co-purifying proteins were identified by 2-DE in combination with MALDI-TOF-MS. Non-liganded/non-activated and in vitro liganded/activated GR, respectively, co-purifies with specific sets of proteins. Of these 34 were conclusively identified, seven have previously been reported to be part of the GR-complex, revealing 27 new possible interacting candidates for the GR-complex. Of the novel GR interacting proteins the major vault protein, TATA binding interacting protein 49a and glycoprotein PP63 were of special interest. Furthermore, using 2-D DIGE we show that the set of proteins interacting with non-liganded GR is distinctly different in protein amount compared to the proteins found with liganded/activated GR. This suggests the presence of different GR complexes in the cell, which was further substantiated by the finding of several separate GR native protein complexes, "GR-receptosomes", using blue native gel electrophoresis. Our findings suggest the existence of several new mechanisms for GR signaling and regulation.

Animals↗
PubMedDate not supplied

In vivo dual-tagging proteomic approach in studying signaling pathways in immune response.

Up to date, few successes have been achieved to identify the signaling molecules directly from immune cells due to their low-abundance and dynamic nature. Here, we designed an in vivo dual-tagging quantitative approach that integrated epitope-tagging which allows single affinity purification of the natural complexes formed at real-time, and amino acid-coded mass tagging (AACT) that assists mass spectrometry-based quantitative measurement, to identify the specific components of a signaling complex formed in macrophage cells upon lipopolysaccharide (LPS) stimulation. The sensitivity and accuracy of this quantitative method are significantly higher than those of tandem affinity purification, because the multiple step of purifications are avoided to preserve weakly interacting molecules. We identified a number of proteins that interact with MyD88, a critical adaptor protein in innate immune response, in macrophages upon stimulation. Among those newly identified MyD88-interacting partners, FLAP-1 was found to be an activator of NF-kappaB, the key transcription factor in immune response. This integrated approach provides global information on the functional link between MyD88 and other proteins in transducing the TLR-mediated signal and is generally applicable to in vivo analyses of other signaling pathways.

5-Lipoxygenase-Activating Proteins↗
PubMed2001

Proteomic analysis of Fasciola hepatica excretory-secretory products.

This paper describes a global investigation of the components of Fasciola hepatica excretory-secretory (ES) products by a proteomic approach. Despite the absence of a F. hepatica genome sequencing project we have shown that it was possible to identify 29 of the 60 prominent proteins found using two-dimensional gel electrophoresis. As well as cathepsin L proteases, a number of enzymes implicated in parasite protection from the host immune system were also found to be present in relatively large abundance. These included superoxide dismutase, thioredoxin peroxidase, glutathione S-transferases and fatty acid binding proteins, all of which may play a part in the detoxification of reactive oxygen intermediates. Interestingly, ovine superoxide dismutase was the only protein from the host identified on the gel. We suggest that the relative abundance and protective nature of the components of the ES products of this organism play an important role in its survival within the host. The precise identification, to individual NCBI database entries, of a number of glutathione S-transferases and cathepsin Ls from F. hepatica, by peptide mass fingerprinting, was hampered by multi-database submissions of the two protein superfamilies from this organism.

Amino Acid Sequence↗