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An intron with a constitutive transport element is retained in a Tap messenger RNA.

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.

Alternative Splicing↗

Characterization of open reading frame-expressed sequence tags generated from Bos indicus and B. taurus mammary gland cDNA libraries.

Sequence-based gene expression data are used to interpret results from functional genomic and proteomics studies. Although more than 300 000 bovine-expressed sequence tags (ESTs) are available in public databases, a more thorough and directed sampling of the expressed genome is needed to identify new transcripts and improve assembly and annotation of existing transcript sequences. Accordingly, we examined the utility of constructing cDNA libraries synthesized by arbitrarily primed RT-PCR of mRNA from tissues not well represented in the publicly available bovine EST database. A total of 33 cDNA libraries were constructed from healthy and infected mammary gland tissues of Brazilian Gir and Holstein cattle. This series of libraries was used to generate 6481 open reading frame-expressed sequence tags (ORESTES) that assembled into 1798 unique sequence elements of which, 1157 did not significantly match sequence assemblies available in the Bos taurus gene index. However, a total of 264 of these 1157 sequence elements aligned with mouse and human expressed sequences demonstrating that ORESTES is an effective resource for discovery of novel expressed sequences in cattle. Furthermore, comparison of the alignment position of bovine ORESTES-derived sequence elements to human gene reference sequences suggested that the priming events for cDNA synthesis more often occurred at the central portion of a transcript, which may have contributed to the relatively high rate of novel sequence discovery.

Animals↗

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA.

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.

Amino Acid Sequence↗

Applications and current challenges of proteomic approaches, focusing on two-dimensional electrophoresis.

Since the formulation of the concept of "proteomics" in 1995, a plethora of proteomic technologies have been developed in order to study proteomes of tissues, cells and organelles. The powerful new technologies enabled by proteomic approaches have lead to the application of these methods to an exponentially increasing variety of biological questions for highly complex protein mixtures. Continuous technical optimization allows for an ever-increasing sensitivity of proteomic techniques. In this review, a brief overview of currently available proteomic techniques and their applications is given, followed by a more detailed description of advantages and technical challenges of two-dimensional electrophoresis (2-DE). Some solutions to circumvent currently encountered technical difficulties for 2-DE analyses are proposed.

Animals↗

Proteomic analysis of apical microvillous membranes of syncytiotrophoblast cells reveals a high degree of similarity with lipid rafts.

Brush borders (microvilli) are cell membrane specialized structures that function mainly as high-throughput absortive/secretory areas. It has been well-established that brush borders are particularly rich in membrane lipids characteristic to lipid rafts. Here, we report 57 proteins identified from microvillous membranes (MVM) isolated from human syncytiotrophoblast cells using an experimental method that avoids the use of nonionic detergents. About 60% of the proteins reported here have been described previously as lipid-raft specific. Well-known lipid raft-markers such as Annexin A2 and alkaline phosphatase were identified. Cytoskeleton structural constituents and proteins related with the control and modulation of the cytoskeletal architecture as well as the regulation of the interaction of cytoskeletal constituents with the cell membrane and particularly with lipid raft domains were found (Ezrin, IQGAP1 and 2, EBP50). Other proteins identified include signal transduction molecules, such as Ras-related protein Rab-1B and Rab-7, and ADP-ribosylation factor 1. Several proteins harbor putative post-translational modifications that favor its localization in the lipid-raft environment, such as GPI (alkaline phosphatase and 5'-nucleotidase) and myristoylation (BASP1 and MARCKS). On the whole, this extensive description demonstrates from the protein composition point of view that brush border membranes are indeed highly enriched in lipid raft microdomains.

Blotting, Western↗

Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis.

Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.

Animals↗

Phylomat: an automated protein motif analysis tool for phylogenomics.

Recent progress in genomics, proteomics, and bioinformatics enables unprecedented opportunities to examine the evolutionary history of molecular, cellular, and developmental pathways through phylogenomics. Accordingly, we have developed a motif analysis tool for phylogenomics (Phylomat, http://alg.ncsa.uiuc.edu/pmat) that scans predicted proteome sets for proteins containing highly conserved amino acid motifs or domains for in silico analysis of the evolutionary history of these motifs/domains. Phylomat enables the user to download results as full protein or extracted motif/domain sequences from each protein. Tables containing the percent distribution of a motif/domain in organisms normalized to proteome size are displayed. Phylomat can also align the set of full protein or extracted motif/domain sequences and predict a neighbor-joining tree from relative sequence similarity. Together, Phylomat serves as a user-friendly data-mining tool for the phylogenomic analysis of conserved sequence motifs/domains in annotated proteomes from the three domains of life.

Algorithms↗

Human Plasma PeptideAtlas.

Peptide identifications of high probability from 28 LC-MS/MS human serum and plasma experiments from eight different laboratories, carried out in the context of the HUPO Plasma Proteome Project, were combined and mapped to the EnsEMBL human genome. The 6929 distinct observed peptides were mapped to approximately 960 different proteins. The resulting compendium of peptides and their associated samples, proteins, and genes is made publicly available as a reference for future research on human plasma.

Blood Proteins↗

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408.

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.

Animals↗

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster.

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.

Animals↗

The human erythrocyte proteome: analysis by ion trap mass spectrometry.

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.

Cell Fractionation↗

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D.

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.

Cell Line, Transformed↗

FindTarget: software for subtractive genome analysis.

In silico subtractive/differential genome analysis is a powerful approach for identifying genus- or species-specific genes, or groups of genes that are responsible for a unique phenotype. By this method, one searches for genes present in one group of bacteria and absent in another group. A software package has been developed, named FindTarget, that has a user-friendly web interface to facilitate differential genome analysis. The user chooses the genomes to compare, the similarity criteria and the thresholds to decide if a gene has a counterpart in another genome. The searches are based on BLASTP comparisons of proteomes. FindTarget also includes access to sequences, coloured multiple alignments, phylogenetic trees of conserved proteins and links to public annotated databases which provide a means for validation of the results. To illustrate this approach, a FindTarget search for genes putatively involved in the specificity of cell envelope synthesis of Gram-negative bacteria is presented. The results show that most of the identified genes are clearly involved in cell wall processes, underlining the power of such an approach in general and that of FindTarget in particular.

Bacterial Proteins↗

On the way to functional genomics.

Proteomics, the global comprehensive analysis of cellular proteins, will contribute to our understanding of gene function in the post-genomic era. The strategies of proteome analysis include the protein expression proteomics dealing with the comparison of cellular or tissue protein levels in the control and affected state (e.g. in the disease) and cell mapping proteomics aimed to define protein-protein interactions that constitute intracellular signalling network. The currently used methods of proteome analysis represent two-dimensional gel electrophoresis for protein separation, the image analysis means for protein maps comparison and the mass spectrometry (MALDI-TOF, MALDI-ESI) and bioinformatics means (sequence databases) for protein identification. For the protein interaction studies the yeast two-hybrid system is mostly employed. New concepts of disease diagnosis and treatment as well as new drugs design can be envisioned from the proteomic analysis.

Genomics↗

hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes.

Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at ). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.

Animals↗

Proteomic analysis of Caco-2 cells treated with monacolin K.

Monascus species is an important traditional fermentation fungus used on food. Monacolin K (a secondary metabolite of Monascus, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase) in inhibition of mevalonate synthesis may result in reductions of isoprenoid prenylation in cells. Impairment of protein isoprenoid prenylation has been related to anticancer effect in cancer cells. As a functional food for Monascus, however, the molecular mechanisms responsible for the anti-proliferate effect of monacolin K in cancer cells are not clear. We used proteomic analysis by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), tandem mass spectrometry (MS/MS), and database interrogation to separate and identify the proteins of Caco-2 cells treated with monacolin K. The results showed that monacolin K inhibited the proliferation of Caco-2 cells in a dose-dependent manner. The identified proteins in proteomic analysis included anti-oxidation enzymes related to reactive oxygen species stress, cytoskeleton proteins, glycolytic enzymes, and enzymes involved in mediating protein interactions. Furthermore, glutathione S-transferase P 1 and cytoskeleton-8, -18, and -19 revealed a down-regulation in a dose-dependent manner in exposure of Caco-2 cells to monacolin K.

Antineoplastic Agents↗

Influence of nicotine on protein expression around hydrophilic osseointegrated implants: A proteomic study in male rats.

OBJECTIVE: To ensure the success of dental implant treatment, various factors must be considered, including osseointegration and systemic conditions. There is evidence in the literature that smokers may exhibit alterations in tissue healing, which can compromise the success of implant rehabilitation. Therefore, this study aimed to investigate the influence of nicotine on the protein profile of bone tissue around hydrophilic implants during the osseointegration process in rats. DESIGN: Bone tissue samples from the control and nicotine groups (n = 3 per group) were subjected to protein extraction, mass spectrometry, and bioinformatic analyses. Protein identification was performed using Proteome Discoverer 2.1 software and the SEQUEST algorithm, and the protein data were compared with those of a protein database of Rattus norvegicus obtained from UniProt. RESULTS: A total of 740 proteins were detected in both the control group and the nicotine-exposed group. Among them, the proteins biglycan, periostin and histone H4 were highlighted because of their higher abundance in the healthy implant group, while they were reduced in the nicotine-exposed group. CONCLUSIONS: Nicotine has the potential to alter the protein profile of bone tissue around hydrophilic implants during osseointegration, which may impair tissue remodeling and healing.

Animals↗

A proteomic view of cell physiology of Bacillus subtilis--bringing the genome sequence to life.

The genome sequence is the "blue-print of life", and the proteomic approach brings this genome sequence to life. Simple model systems are urgently required to "train" this transformation of the genome sequence into life: why not Bacillus subtilis, the model organism for gram-positive bacteria and of functional genomics? By combination of the highly sensitive 2D protein gel electrophoresis with the identification of the protein spots by microsequencing or mass spectrometry we established a 2D protein index of Bacillus subtilis. In order to depict the entire proteome of a B. subtilis cell, alkaline, cell-wall associated, or extracellular proteins were also included. The proteins of this database (see http://microbio2.biologie.uni-greifswald.de:8880/sub2d.htm) were allocated to proteins with house-keeping functions typical of growing cells and to proteins synthesized particularly in non-growing cells. A computer-aided evaluation of the 2D gels loaded with radioactively-labeled proteins from growing or stressed/starved cells proved to be a powerful tool for the analysis of global regulation of the expression of the entire genome. This is shown for the analysis of glycolysis/TCA cycle (house keeping proteins) and for the analysis of the heat stress stimulon. For the heat stress stimulon it is demonstrated how the proteomic approach can be used: (i) to define the structure of a stimulon, (ii) to dissect stimulons into regulons, (iii) to analyze the regulation, structure, and function of unknown regulons, (iv) to define overlapping reguIons or modulons, and finally (v) to explore complex adaptational networks. Furthermore, it will be demonstrated how the "dual channel pattern comparison" or "proteomics signature" (R. VanBogelen) can be used for a comprehensive understanding or prediction of the physiological state of growing or starving cell populations. This is shown for glucose-starved cells. In order to describe the structure and function of gene regulation groups it is generally recommended to complement the proteomics approach with DNA array technologies. Further studies will focus on the analysis of the global regulation of gene expression by the proteomic approach that cannot be addressed by the application of DNA array techniques: the phosphoproteome and its implications in signal transduction; the global control of protein stability; protein targeting and protein secretion.

Adaptation, Physiological↗