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Results for “proteome mapping”

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PubMed2003

Analysis of protein interactions using fluorescence technologies.

Biophotonics techniques, especially those involving fluorescence, are widely used in proteomics to characterize the in vitro interactions between proteins in high-throughput mode. On the other hand, fluorescence-based imaging studies often show that protein activity is regulated through large protein complexes that transiently form at specific sites in the cell. One could therefore argue that a systematic functional analysis of the human proteome requires technologies that are capable of time and spatially resolved, multiplexed analysis of protein interactions within cells.

Fluorescence Resonance Energy Transfer↗

PubMed2006

Improvements in the search for potential biomarkers by proteomics: application of principal component and discriminant analyses for two-dimensional maps evaluation.

In this study, we evaluated if the application of multivariate analysis on the data obtained from two-dimensional protein maps could mean an improvement in the search for protein markers. First, we performed a classical proteomic study of the differential expression of serum N-glycoproteins in colorectal cancer patients. Then, applying principal component analysis (PCA) we assessed the utility of the 2-D protein pattern and certain subsets of spots as a tool to distinguish control and case samples, and tested the accuracy of the classification model by linear discriminant analysis (LDA). On the other hand we looked for altered spots by univariate statistics and then analysed them as a cluster by PCA and LDA. We found that those proteins combined presented a theoretical sensitivity and specificity of 100%. Finally, the spots with known protein identity were analysed by multivariate methods, finding a subgroup that behaved as the most obvious candidates for further validation trials.

Biomarkers↗

PubMed2003

Proteome analysis at the level of subcellular structures.

The targeting of proteins to particular subcellular sites is an important principle of the functional organization of cells at the molecular level. In turn, knowledge about the subcellular localization of a protein is a characteristic that may provide a hint as to the function of the protein. The combination of classic biochemical fractionation techniques for the enrichment of particular subcellular structures with the large-scale identification of proteins by mass spectrometry and bioinformatics provides a powerful strategy that interfaces cell biology and proteomics, and thus is termed 'subcellular proteomics'. In addition to its exceptional power for the identification of previously unknown gene products, the analysis of proteins at the subcellular level is the basis for monitoring important aspects of dynamic changes in the proteome such as protein transloction. This review summarizes data from recent subcellular proteomics studies with an emphasis on the type of data that can retrieved from such studies depending on the design of the analytical strategy.

Animals↗

PubMed2004

Polyanions and the proteome.

The behavior of the proteome reflects spatial and temporal organization both within and without cells. We propose that various macromolecular entities possessing polyanionic character such as proteoglycans, lipid bilayer surfaces, microtubules, microfilaments, and polynucleotides may provide a functional network that mediates a variety of cellular phenomena. The interaction of proteins with this array of polyanions is characterized by a lower degree of specificity than seen with most commonly recognized macromolecular interactions. In this commentary, potential roles for this polyanion network in diverse functions such as protein/protein interactions, protein folding and stabilization, macromolecular transport, and various disease processes are all considered, as well as the use of polyanions as therapeutic agents. The role of small polyanions in the regulation of protein/polyanion interactions is also postulated. We provide preliminary experimental analysis of the extent to which proteins interact with polyanions inside cells using a combination of two-dimensional chromatographic and electrophoretic methods and antibody arrays. We conclude that many hundreds to thousands of such interactions are present in cells and argue that future understanding of the proteome will require that the "polyanion world" be taken into account.

Animals↗

PubMed2001

Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y neuroblastoma cell line.

To probe the mitochondrial involvement in neurodegenerative processes, we have generated a high-resolution map of the mitochondrial proteome from a human neuroblastoma SH-SY5Y cell line that has been used for creating cytoplasmic hybrid cell systems. Two mitochondrial preparations were evaluated using two-dimensional (2D) gel electrophoresis and mass spectrometry; one obtained from differential centrifugation and the other by a multiple-step percoll/metrizamide gradient. The 2D gel maps prepared from these mitochondrial fractions separated over 300 distinct spots as visualized by colloidal Coomassie blue (CCB), or closer to 400 proteins with silver staining. The most abundant proteins identified in the mitochondrial fraction prepared by differential centrifugation were those of mitochondrial, cytoplasmic, and endoplasmic reticulum origin. Proteins obtained using the more intensive two-step gradient method were almost exclusively known to be associated with mitochondria. From this latter preparation, 84 of the most abundant gel spots were analyzed, out of which 61 proteins were identified. The absence of many membrane-associated proteins known to be associated with the mitochondrion and the limited number of total proteins observed in the 2D gel maps suggest that the majority of mitochondrial proteins are not being detected under these separation and staining conditions. An insoluble pellet obtained after solubilization of the mitochondrial fraction prepared with the percoll/metrizamide gradient was boiled in sodium dodecylsulfate (SDS) and separated by 1D sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This separation yielded some additional proteins, many of which are likely membrane-associated. These studies form the basis for the analysis of differential protein expression in cybrid cellular models of neurodegenerative disorders and in affected tissue from diseased states.

Journal Article↗

PubMed2004

Quantitative genomics: exploring the genetic architecture of complex trait predisposition.

Most phenotypes with agricultural or biomedical relevance are multifactorial traits controlled by complex contributions of genetics and environment. Genetic predisposition results from combinations of relatively small effects due to variations within a large number of genes, known as QTL. Well over 200 QTL have been reported for growth and body composition traits in the mouse, which likely represent at least 50 to 100 distinct genes. Molecular biology has yielded significant advances in understanding these traits at the metabolic and physiological levels; however, little has been learned regarding the identity and nature of the underlying polygenes. In addition to the significantly poor precision inherent to QTL localization, it is very difficult to differentiate between co-localization and coincidence when comparing QTL with other QTL and with potential candidate genes. The wide gap between our knowledge of physiological mechanisms underlying complex traits and the nature of genetic predisposition significantly impairs discovery of genes underlying QTL. Identification and genetic mapping of key transcriptional, proteomic, metabolomic, and endocrine events will uncover large lists of significant positional candidate genes for growth and body composition. However, integration of experimental approaches to jointly evaluate predisposition and physiology will increase success of QTL identification by merging the power of recombination with functional analysis. Measuring physiologically relevant subphenotypes within a structured QTL mapping population will not only facilitate pathway-specific prioritization among candidate genes, but may also directly identify genes underlying QTL. This would advance our understanding of the genetic architecture of complex traits by testing the central hypothesis that genes controlling predisposition to a quantitative trait are primarily involved in trans-regulation of the primary physiological pathways that regulate the trait.

Animals↗

PubMedDate not supplied

Global differential non-gel proteomics by quantitative and stable labeling of tryptic peptides with oxygen-18.

We describe a protocol for quantitative labeling of tryptic peptides with oxygen-18. Proteins are first digested in natural water with trypsin, the pH is then lowered to 4.5 and the mixture is dried. Oxygen-18 water is added and two oxygen-18 atoms are incorporated at the peptides' carboxyl termini. Trypsin is finally inactivated by cysteine alkylation under denaturing conditions, which blocks oxygen back-exchange. The general value of this labeling strategy for differential proteomics is illustrated by the analysis and identification of several couples of differently labeled amino terminal peptides isolated from a human platelet proteome by a previously described chromatographic procedure.

Amino Acid Sequence↗

PubMed2004

Quantitative proteome analysis in benign thyroid nodular disease using the fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain.

Thyroid tumorigenesis involves qualitative and quantitative changes in protein expression, which can be comprehensively studied by proteome analysis. However, one of the technical bottlenecks of proteomics remains a reliable, sensitive and inexpensive method for quantification of differentially expressed proteins. This is due to the limited linear range of most available protein stains, i.e. silver and Coomassie blue, and high costs of commercially available fluorescent stains. In this paper we describe our experience with a lab-made ruthenium based fluorescent stain (ruthenium II tris(bathophenanthroline disulfonate) (RuBPs)) to perform proteome analysis of nodular thyroid disease. We first compared the properties of RuBPs with two highly sensitive protein stains: (1) silver staining and (2) the commercially available fluorescent dye Sypro Ruby. We show that in addition to its highly sensitive staining capabilities similar to Sypro Ruby and silver (2 ng), RuBPs offers several advantages such as a broad dynamic range (similar to Sypro Ruby and 500 times broader than the dynamic range of silver stain), low costs ( 0.03 per gel) and excellent compatibility with mass spectrometry. We then applied the inexpensive RuBPs stain to 2D gels (pH 4-7) of four benign thyroid nodules and normal thyroid tissue. We were able to detect approximately 1800 protein spots/gel in our thyroid samples. Quantitative changes in protein expression levels of at least 20-42 proteins were noted in the benign nodules compared with the normal thyroid tissue of the same patient. Differentially expressed spots were further characterised by nano-LC-FTICR and MALDI-TOF mass spectrometry. In summary we demonstrate, that the novel fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain is a highly sensitive, reliable and inexpensive tool for quantitative proteome analysis in thyroid nodular disease.

Dextrans↗

PubMed2004

Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates.

Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients. Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not. Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins. About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity. This percentage reflects a relatively high degree of intraclonal variability. Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors. It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

Clone Cells↗

PubMed2001

Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes.

To pass through its life cycle, protozoan parasites of the genus Leishmania have to differentiate from promastigotes to amastigotes. The molecular basis underiving this major transformation is poorly understood. One way to study this phenomenon is to isolate and characterize proteins that are specifically expressed in one of the two stages of the life cycle or during the stage differentiation. Using two-dimensional gel electrophoresis, we mapped the Leishmania donovani proteome during stage differentiation to identify stage-specific proteins and regulons. A protocol for extracting proteins of both promastigote and amastigote L. donovani cells was developed, which is compatible with isoelectric focusing. Up to 400 L. donovani protein spots were visualized on a silver-stained gel. Metabolic labeling of the cells was used to compare directly the protein synthesis pattern with the protein level pattern. The silver-stained images of L. donovani cells harvested on different days of stage differentiation were compared to the corresponding autoradiographs. A marked decrease in protein synthesis during stage differentiation from promastigotes to amastigotes was observed. The stained protein pattern as well as the protein pattern on the autoradiograph changed dramatically, especially after day 3 (about 24 h after pH shift) of transformation.

Animals↗

PubMed2000

Protein analysis by mass spectrometry and sequence database searching: a proteomic approach to identify human lymphoblastoid cell line proteins.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

Acrylic Resins↗

PubMed2006

Proteome profiling in the rat Harderian gland.

The Harderian gland is an orbital gland located behind the ocular bulb in most terrestrial vertebrates probably functioning for production of lipid secretion to protect the eye. We herein present a protein reference database of the rat Harderian gland that may serve as analytical tool for future proteomic work, report lipid and porphyrin handling cascades, address sequence conflicts and report structures that have not been so far described by proteomics methods.

Amino Acid Sequence↗

PubMed2026

Emerging Principles in Spatial Functional Genomics.

Spatial transcriptomic and proteomic atlases have enabled mapping of gene programs within intact tissues, but these measurements remain largely descriptive and do not define the mechanisms controlling tissue biology. Pooled CRISPR screening provides scalable causal interrogation of gene function but remains largely confined to dissociated systems that lack spatial context. In vivo spatial functional genomics (SFG) bridges these approaches by integrating genetic perturbations with in situ transcriptomic and proteomic readouts to measure gene function within intact tissue ecosystems. By preserving spatial organization, SFG enables interpretation of perturbations through effects on cell-cell interactions, diffusible signals, multicellular niches, and tissue architecture. Here, we outline key design axes of SFG: perturbation strategy, barcoding strategy, and phenotypic readout. We discuss computational challenges, including spatial autocorrelation, neighborhood dependence, and context-aware null modeling, and highlight how SFG reveals non-cell-autonomous, architecture-dependent mechanisms of gene function, advancing toward predictive models of tissue organization and gene function.

Genomics↗

PubMed2004

Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis.

Separation of proteins by two-dimensional electrophoresis and following mass spectrometry (MS) is now a conventional technique for proteomic analysis. For proteomic analysis of a certain tissue with a limited information of primary structures of proteins, we have developed an analytical system for peptide mass fingerprinting in gene products in the testis of the ascidian Ciona intestinalis. Ciona sperm proteins were separated by two-dimensional gel electrophoresis and the tryptic fragments were subjected to MALDI-TOF/MS. The mass pattern was searched against on-line databases but resulted in less identification of these proteins. We have constructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied for proteomic analysis for a limited tissue with less genomic information.

Animals↗

PubMed2006

Mass spectrometry-based proteomics combined with bioinformatic tools for bacterial classification.

Timely classification and identification of bacteria is of vital importance in many areas of public health. We present a mass spectrometry (MS)-based proteomics approach for bacterial classification. In this method, a bacterial proteome database is derived from all potential protein coding open reading frames (ORFs) found in 170 fully sequenced bacterial genomes. Amino acid sequences of tryptic peptides obtained by LC-ESI MS/MS analysis of the digest of bacterial cell extracts are assigned to individual bacterial proteomes in the database. Phylogenetic profiles of these peptides are used to create a matrix of sequence-to-bacterium assignments. These matrixes, viewed as specific assignment bitmaps, are analyzed using statistical tools to reveal the relatedness between a test bacterial sample and the microorganism database. It is shown that, if a sufficient amount of sequence information is obtained from the MS/MS experiments, a bacterial sample can be classified to a strain level by using this proteomics method, leading to its positive identification.

Amino Acid Sequence↗

PubMedDate not supplied

STEM: a software tool for large-scale proteomic data analyses.

We describe the software, STEM (STrategic Extractor for Mascot's results), which efficiently processes large-scale mass spectrometry-based proteomics data. V (View)-mode evaluates the Mascot peptide identification dataset, removes unreliable candidates and redundant assignments, and integrates the results with key information in the experiment. C (Comparison)-mode compares peptide coverage among multiple datasets and displays proteins commonly/specifically found therein, and processes data for quantitative studies that utilize conventional isotope tags or tags having a smaller mass difference. STEM significantly improves throughput of proteomics study.

Algorithms↗

PubMed2004

Tandem affinity purification of the Candida albicans septin protein complex.

A novel vector was constructed to enable the integrative marking of individual genes and the affinity purification of interacting molecules within protein complexes from Candida albicans using a tandem 6 x histidine and FLAG epitope tag. The system was verified by purifying the C. albicans septin complex (a self-associating complex of cytoskeletal proteins) from both yeast and hyphal cells. One-step affinity purification was insufficient for purification of the protein complex, whereas tandem affinity purification (TAP) gave an extensively purified protein complex with a very low background. Electrophoretic and mass spectrometry analysis showed that the affinity-purified C. albicans septin complex, which comprises predominantly CaCdc3p, CaCdc10p, CaCdc11p, CaCdc12p and CaSep7p, was not affected by cell morphology. The purified septin complex appeared to have a stoichiometry of 2 CaCdc3p, 1-2 CaCdc10p, 1 CaCdc11p, 2 CaCdc12p and < or = 1 CaSep7p. The successful application of TAP to the purification and analysis of the C. albicans septin complex indicates that this technology will have much wider application to proteomic studies of this pathogenic fungus.

Amino Acid Sequence↗

PubMed2002

Prediction, assessment and validation of protein interaction maps in bacteria.

High-throughput proteomics technologies, especially the yeast two-hybrid system, produce large volumes of protein-protein interaction data organized in networks. The complete sequencing of many genomes raises questions about the extent to which such networks can be transferred between organisms. We attempted to answer this question using the experimentally derived Helicobacter pylori interaction map and the recently described interacting domain profile pair (IDPP) method to predict a virtual map for Escherichia coli. The extensive literature concerning E.coli was used to assess all predicted interactions and to validate the IDPP method, which clusters protein domains by sequence and connectivity similarities. The IDPP method has a much better heuristic value than methods solely based on protein homology. The IDPP method was further applied to Campylobacter jejuni to generate a virtual interaction map. An in-depth comparison of the chemotaxis pathways predicted in E.coli and C.jejuni led to the proposition of new functional assignments. Finally, the prediction of protein-protein interaction maps across organisms enabled us to validate some of the interactions on the original experimental map.

Bacterial Proteins↗