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Results for “proteome mapping”

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PubMed2006

Serum, liver, and kidney proteomic analysis for the alloxan-induced type I diabetic mice after insulin gene transfer of naked plasmid through electroporation.

Gene therapy has been reported to be effective in treating diabetes mellitus (DM), while little has been found out about the functional protein changes since. The liver and kidney play important roles in glucose absorption, metabolism, and excretion. Changes in the two organs may reflect pathologic alterations during DM, while the serum has a direct connection with most organs and pathological changes. We used alloxan to induce diabetic mice, electrotranferred the insulin gene into their sural muscles, and discovered that their blood glucose decreased to normal level. Consequently, proteomic approaches were applied to evaluate protein changes in the liver, kidney, and serum of normal, diabetic, and gene transferred mice. Forty-three proteins were found either up-regulated or down-reglulated in the liver, kidney, and serum of the alloxan-induced type I diabetic mice. Only five proteins in the liver, five proteins in the kidney, and seven proteins in the serum of diabetic mice were found to be back-regulated to normal levels after gene transfer. These back-regulated proteins are involved in lipid and glucose metabolism, associated with phosphorylation, signal transduction, oxidation, and immune inflammation. Our findings might promote a better understanding for the mechanism of DM, and provide novel targets for estimating the effects of gene therapy.

Animals↗

PubMed2006

Proteomic analysis of the mouse mammary gland is a powerful tool to identify novel proteins that are differentially expressed during mammary development.

After lactation, the mouse mammary gland undergoes apoptosis and tissue remodelling as the gland reverts to its prepregnant state. This complex change was investigated using 2-DE. An integrated database was produced from lactation and involution proteomes. Forty-four molecular cluster indexes (MCIs) that showed altered expression from lactation to involution were selected for MS analysis. Of these, 32 gave protein annotations, 18 of which were unequivocal proteins. Selected proteins were then studied across all of development, including pregnancy, using data integrated from another proteome database. Two proteins, the RNA polymerase B transcription factor 3 (BTF3) and the minichromosome maintenance protein 3 (MCM3), although initially selected on the basis of the lactation/involution criteria, had expression profiles that indicated an additional role in mammary development and were further analysed. BTF3, a transcription factor previously not described in the mammary gland, was up-regulated strongly in pregnancy, indicating an involvement in alveolar growth. MCM3's expression was greatest in pregnancy and late involution, decreasing through lactation. Immunohistochemistry localised MCM3 to the mammary epithelium, where a greater proportion of cells stained than for the proliferation marker Ki67. MCM3 expression during lactation may identify cells that are licensed to repopulate the gland during cell loss in lactation and following involution.

Animals↗

PubMed2004

Phenotype characterisation using integrated gene transcript, protein and metabolite profiling.

Multifactorial diseases present a significant challenge for functional genomics. Owing to their multiple compartmental effects and complex biomolecular activities, such diseases cannot be adequately characterised by changes in single components, nor can pathophysiological changes be understood by observing gene transcripts alone. Instead, a pattern of subtle changes is observed in multifactorial diseases across multiple tissues and organs with complex associations between corresponding gene, protein and metabolite levels. This article presents methods for exploratory and integrative analysis of pathophysiological changes at the biomolecular level. In particular, novel approaches are introduced for the following challenges: (i) data processing and analysis methods for proteomic and metabolomic data obtained by electrospray ionisation (ESI) liquid chromatography-tandem mass spectrometry (LC/MS); (ii) association analysis of integrated gene, protein and metabolite patterns that are most descriptive of pathophysiological changes; and (iii) interpretation of results obtained from association analyses in the context of known biological processes. These novel approaches are illustrated with the apolipoprotein E3-Leiden transgenic mouse model, a commonly used model of atherosclerosis. We seek to gain insight into the early responses of disease onset and progression by determining and identifying--well in advance of pathogenic manifestations of disease--the sets of gene transcripts, proteins and metabolites, along with their putative relationships in the transgenic model and associated wild-type cohort. Our results corroborate previous findings and extend predictions for three processes in atherosclerosis: aberrant lipid metabolism, inflammation, and tissue development and maintenance.

Algorithms↗

PubMed2003

Proteomic approaches to the diagnosis, treatment, and monitoring of cancer.

The field of proteomics holds promise for the discovery of new biomarkers for the early detection and diagnosis of disease, molecular targets for therapy and markers for therapeutic efficacy and toxicity. A variety of proteomics approaches may be used to address these goals. Two-dimensional gel electrophoresis (2D-PAGE) is the cornerstone of many discovery-based proteomics studies. Technologies such as laser capture microdissection (LCM) and highly sensitive MS methods are currently being used together to identify greater numbers of lower abundance proteins that are differentially expressed between defined cell populations. Newer technologies such as reverse phase protein arrays will enable the identification and profiling of target pathways in small biopsy specimens. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) analysis enables the high throughput characterization of lysates from very few tumor cells or body fluids and may be best suited for diagnosis and monitoring of disease. Such technologies are expected to supplement our arsenal of mRNA-based assays, and we believe that in the future, entire cellular networks and not just a single deregulated protein will be the target of therapeutics and that we will soon be able to monitor the status of these pathways in diseased cells before, during and after therapy.

Biomarkers, Tumor↗

PubMed2006

Universal metrics for quality assessment of protein identifications by mass spectrometry.

Increasing numbers of large proteomic datasets are becoming available. As attempts are made to interpret these datasets and integrate them with other forms of genomic data, researchers are becoming more aware of the importance of data quality with respect to protein identification. We present three simple and universal metrics that describe different aspects of the quality of protein identifications by peptide mass fingerprinting. Hit ratio gives an indication of the signal-to-noise ratio in a mass spectrum, mass coverage measures the amount of protein sequence matched, and excess of limit-digested peptides reflects the completeness of the digestion that precedes the peptide mass fingerprinting. Receiver-operating characteristic plots show that the novel metric, excess of limit-digested peptides, can discriminate between correct and random matches more accurately than search score when validating the results from a state-of-the-art protein identification software system (Mascot) especially when combined with the two other metrics, hit ratio and mass coverage. Recommendations are made regarding the use of the metrics when reporting protein identification experiments.

Mass Spectrometry↗

PubMed2004

The HUPO PSI's molecular interaction format--a community standard for the representation of protein interaction data.

A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).

Database Management Systems↗

PubMed2004

Automating proteome analysis: improvements in throughput, quality and accuracy of protein identification by peptide mass fingerprinting.

The use of robots has major effects on maximizing the proteomic workflow required in an increasing number of high-throughput projects and on increasing the quality of the data. In peptide mass finger printing (PMF), automation of steps downstream of two-dimensional gel electrophoresis is essential. To achieve this goal, the workflow must be fluid. We have developed tools using macros written in Microsoft Excel and Word to complete the automation of our platform. Additionally, because sample preparation is crucial for identification of proteins by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we optimized a sandwich method usable by any robot for spotting digests on a MALDI target. This procedure enables further efficient automated washing steps directly on the MALDI target. The success rate of PMF identification was evaluated for the automated sandwich method, and for the dried-droplet method implemented on the robot as recommended by the manufacturer. Of the two methods, the sandwich method achieved the highest identification success rate and sequence coverage of proteins.

Colonic Neoplasms↗

PubMed2006

Mass spectrometry-based proteomic analysis of the epitope-tag affinity purified protein complexes in eukaryotes.

In recent years, MS has been widely used to study protein complex in eukaryotes. The identification of interacting proteins of a particular target protein may help defining protein-protein interaction and proteins of unknown functions. To isolate protein complexes, high-speed ultracentrifugation, sucrose density-gradient centrifugation, and coimmunoprecipitation have been widely used. However, the probability of getting nonspecific binding is comparatively high. Alternatively, by use of one- or two-step (tandem affinity purification) epitope-tag affinity purification, protein complexes can be isolated by affinity or immunoaffinity columns. These epitope-tags include protein A, hexahistidine (His), c-Myc, hemaglutinin (HA), calmodulin-binding protein, FLAG, maltose-binding protein, Strep, etc. The isolated protein complex can then be subjected to protease (i.e., trypsin) digestion followed by an MS analysis for protein identification. An example, the epitope-tag purification of the Arabidopsis cytosolic ribosomes, is addressed in this article to show the success of the application. Several representative protein complexes in eukaryotes been isolated and characterized by use of this approach are listed. In this review, the comparison among different tag systems, validation of interacting relationship, and choices of MS analysis method are addressed. The successful rate, advantages, limitations, and challenges of the epitope-tag purification are also discussed.

Affinity Labels↗

PubMed2001

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.

Animals↗

PubMedDate not supplied

Multidimensional proteome analysis of human mammary epithelial cells.

Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with the separation of tryptically digested peptides using a combination of strong cation exchange and high resolution, reversed phase capillary chromatography to identify proteins extracted from human mammary epithelial cells (HMECs). In addition to conventional conservative criteria for protein identifications, the confidence levels were additionally increased through the use of peptide normalized elution times (NET) for the liquid chromatographic separation step. The combined approach resulted in a total of 5838 unique peptides identified covering 1574 different proteins with an estimated 4% gene coverage of the human genome, as annotated by the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.

Cell Line↗

PubMed2006

Proteomic analysis of the tetraspanin web using LC-ESI-MS/MS and MALDI-FTICR-MS.

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of a molecular network of interactions, the "tetraspanin web". Here, we have performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of two cell lines derived from primary tumor and metastasis from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification and the proteins were identified by MS using LC-ESI-MS/MS and MALDI-FTICR. The high resolution and mass accuracy of FTICR MS allowed reliable identification using mass finger printing with only two peptides. Thus, it could be used to resolve the composition of complex peptide mixtures from membrane proteins. Different types of membrane proteins were identified, including adhesion molecules (integrins, Lu/B-CAM, GA733 proteins), receptors and signaling molecules (BAI2, PKC, G proteins), proteases (ADAM10, TADG15), and membrane fusion proteins (syntaxins) as well as poorly characterized proteins (CDCP1, HEM-1, CTL1, and CTL2). Some components were differentially detected in the tetraspanin web of the two cell lines. These differences may be relevant for tumor progression and metastasis.

Amino Acid Sequence↗

PubMed2002

Detection of peptides, proteins, and drugs that selectively interact with protein targets.

Genome sequencing has been completed for multiple organisms, and pilot proteomic analyses reported for yeast and higher eukaryotes. This work has emphasized the facts that proteins are frequently engaged in multiple interactions, and that governance of protein interaction specificity is a primary means of regulating biological systems. In particular, the ability to deconvolute complex protein interaction networks to identify which interactions govern specific signaling pathways requires the generation of biological tools that allow the distinction of critical from noncritical interactions. We report the application of an enhanced Dual Bait two-hybrid system to allow detection and manipulation of highly specific protein-protein interactions. We summarize the use of this system to detect proteins and peptides that target well-defined specific motifs in larger protein structures, to facilitate rapid identification of specific interactors from a pool of putative interacting proteins obtained in a library screen, and to score specific drug-mediated disruption of protein-protein interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates.

This paper describes a procedure for quantitative proteomics that selects peptides containing both cysteine and histidine residues from tryptic digests of cell lysates. Cysteine-containing peptides were selected first by covalent chromatography using thiol disulfide exchange. Following the release of cysteine-containing peptides from the covalent chromatography column with reductive cleavage, histidine-containing peptides were captured by passage through an immobilized metal affinity chromatography column loaded with copper. Quantification was achieved in a four-step process involving (i) differential labeling of control and experimental samples with isotopically differing forms of succinic anhydride, (ii) mixing the two globally labeled samples, (iii) fractionating the labeled peptides by reversed-phase liquid chromatography, and (iv) determining the isotope ratio in individual peptides by mass spectrometry. The results of these studies indicate that by selecting peptides containing both cysteine and histidine, the complexity of protein digests could be substantially reduced. Up-regulated proteins from plasmid bearing Escherichia coli that had been induced with isopropyl beta-thiogalacto-pyranoside were identified and quantified by the global internal standard technology (GIST) described above. Database searches were greatly simplified because the number of possible peptide candidates was reduced more than 95%.

Amino Acid Sequence↗

PubMed2006

A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells.

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.

Animals↗

PubMed2003

Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis.

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.

Adaptation, Physiological↗

PubMedDate not supplied

Visual analysis of gel-free proteome data.

We present a visual exploration system supporting protein analysis when using gel-free data acquisition methods. The data to be analyzed is obtained by coupling liquid chromatography (LC) with mass spectrometry (MS). LC-MS data have the properties of being nonequidistantly distributed in the time dimension (measured by LC) and being scattered in the mass-to-charge ratio dimension (measured by MS). We describe a hierarchical data representation and visualization method for large LC-MS data. Based on this visualization, we have developed a tool that supports various data analysis steps. Our visual tool provides a global understanding of the data, intuitive detection and classification of experimental errors, and extensions to LC-MS/MS, LC/LC-MS, and LC/LC-MS/MS data analysis. Due to the presence of randomly occurring rare isotopes within the same protein molecule, several intensity peaks may be detected that all refer to the same peptide. We have developed methods to unite such intensity peaks. This deisotoping step is visually documented by our system, such that misclassification can be detected intuitively. For differential protein expression analysis, we compute and visualize the differences in protein amounts between experiments. In order to compute the differential expression, the experimental data need to be registered. For registration, we perform a nonrigid warping step based on landmarks. The landmarks can be assigned automatically using protein identification methods. We evaluate our methods by comparing protein analysis with and without our interactive visualization-based exploration tool.

Chromatography, Gel↗

PubMed2003

Matrix-assisted laser desorption/ionization- quadrupole ion trap-time of flight mass spectrometry sequencing resolves structures of unidentified peptides obtained by in-gel tryptic digestion of haptoglobin derivatives from human plasma proteomes.

Two-dimensional gel electrophoresis-separated and excised haptoglobin alpha2-chain protein spots were subjected to in-gel digestion with trypsin. Previously unassigned peptide ion signals observed in mass spectrometric fingerprinting experiments were sequenced using the matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) mass spectrometer and showed that the haptoglobin alpha-chain derivative under study was cleaved by trypsin unspecifically. Abundant cleavages occurred C-terminal to histidine residues at H23, H28, and H87. In addition, mild acidic hydrolysis leading to cleavage after aspartic acid residues at D13 was observed. The uninterpreted tandem mass spectrometry (MS/MS) spectrum of the peptide with ion signal at 2620.19 was submitted to database search and yielded the identification of the corresponding peptide sequence comprising amino acids (aa) aa65-87 from the haptoglobin alpha-chain protein. Also, the presence of a mixture of two tryptic peptides (mass to charge ratio m/z 1708.8; aa40-54, and aa99-113, respectively), that is caused by a tiny sequence variation between the two repeats in the haptoglobin alpha2-chain protein was resolved by MS/MS fragmentation using the MALDI-QIT-TOF mass spectrometer instrument. Advantageous features such as (i) easy parent ion creation, (ii) minimal sample consumption, and (iii) real collision induced dissociation conditions, were combined successfully to determine the amino acid sequences of the previously unassigned peptides. Hence, the novel mass spectrometric sequencing method applied here has proven effective for identification of distinct molecular protein structures.

Alleles↗

PubMed2006

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin.

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.

Aged↗