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At least 1,423 records · Page 79Linked to original sources
PubMed2005

Heat shock protein 27 is associated with freedom from graft vasculopathy after human cardiac transplantation.

Experimental studies have suggested that protective genes protect allografts from cardiac allograft vasculopathy (CAV), the major complication after cardiac transplantation. Here we have sought to confirm this hypothesis using long-term heart transplant recipients. Twenty-two patients that were 9 years or older after transplant were investigated; 11 of these were without angiographic evidence of CAV; 11 had developed early CAV at 1 to 3 years after transplant. To identify proteins that may act as protectors from CAV, a global proteomic approach was used comparing cardiac biopsies from 12 patients taken within the first 2 weeks after transplant and those taken after 9 years from the same patient. Proteins were separated by 2-D gel-electrophoresis, detected by silver staining, and analyzed using Progenesis software. A particular protein spot was found in 4/6 biopsies from patients without CAV, but absent from 5/6 biopsies from those with CAV (P=0.24); however, quantitative analysis of spot intensity showed a significant difference (0.061+/-0.05 versus 0.003+/-0.01, P=0.04). This spot was identified by mass spectrometry and a combination of techniques as a diphosphorylated form of HSP27. Immunohistochemistry of further biopsies not only validated that HSP27 was more abundantly expressed on biopsies without CAV but also showed it to be localized to blood vessels. In contrast, vessels from patients with CAV did not express HSP27 (P=0.028x10(-4)). Immunohistochemistry of 12 further early biopsies and nontransplanted heart showed HSP27 to be present in normal blood vessels. These findings suggest that expression of a specific diphosphorylated form of HSP27 is associated with healthy blood vessels; it appears to be lost from vessels of patients with graft vasculopathy.

Apoptosis↗
PubMed2006

SELDI-TOF-MS of saliva: methodology and pre-treatment effects.

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.

Biomarkers↗
PubMed2006

Recent applications of affinity interactions in capillary electrophoresis.

Systems biology depends on a comprehensive assignment and characterization of the interactions of proteins and polypeptides (functional proteomics) and of other classes of biomolecules in a given organism. High-capacity screening methods are in place for ligand capture and interaction screening, but a detailed dynamic characterization of molecular interactions under physiological conditions in efficiently separated mixtures with minimal sample consumption is presently provided only by electrophoretic interaction analysis in capillaries, affinity CE (ACE). This has been realized in different fields of biology and analytical chemistry, and the resulting advances and uses of ACE during the last 2.5 years are covered in this review. Dealing with anything from small divalent metal ions to large supramolecular assemblies, the applications of ACE span from low-affinity binding of broad specificity being exploited in optimizing selectivity, e.g., in enantiomer analysis to miniaturized affinity technologies, e.g., for fast processing immunoassay. Also, approaches that provide detailed quantitative characterization of analyte-ligand interaction for drug, immunoassay, and aptamer development are increasingly important, but various approaches to ACE are more and more generally applied in biological research. In addition, the present overview emphasizes that distinct challenges regarding sensitivity, parallel processing, information-rich detection, interfacing with MS, analyte recovery, and preparative capabilities remain. This will be addressed by future technological improvements that will ensure continuing new applications of ACE in the years to come.

Animals↗
PubMed2006

Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species.

The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

Aconitate Hydratase↗
PubMed2004

Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni.

Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.

Amino Acid Sequence↗
PubMed2003

JP-8 jet fuel exposure alters protein expression in the lung.

The purpose of this study was to investigate the proteomic mechanisms of Jet Propulsion-8 (JP-8) toxicity in the lung, specifically relating to lung epithelial cell apoptosis and edema. Male Swiss-Webster mice were exposed to 1 h/day aerosolized JP-8 jet fuel at concentrations of 250, 1000, and 2500 mg/m(3) for 7 days. Lung cytosol and whole lung samples were solubilized, separated via large scale, high-resolution two-dimensional electrophoresis, and processed for analysis. Significant quantitative differences in lung protein expression were found as a result of JP-8 exposure. At 250 mg/m(3) JP-8 concentration, 31 proteins exhibited increased expression, while 10 showed decreased expression. At 1000 mg/m(3) exposure levels, 21 lung proteins exhibited increased expression and 99 demonstrated decreased expression. At 2500 mg/m(3), 30 exhibited increased expression, while 135 showed decreased expression. Several of the proteins were identified by peptide mass fingerprinting, and were found to relate to cell structure, cell proliferation, protein repair, and apoptosis. These data demonstrate the significant stress JP-8 jet fuel puts on lung epithelium. Furthermore, there was a decrease in alpha1-anti-trypsin expression suggesting that JP-8 jet fuel exposure may have implications for the development of pulmonary disorders.

Administration, Inhalation↗
PubMed2006

Proteomics analysis revealed changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure.

Exposure to oil mist has been associated with a variety of acute and chronic respiratory effects. Using proteomics approaches to investigate exposure-associated proteins may provide useful information to understand the mechanisms of associated respiratory effects. The aim of this study was to investigate changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure using nano-HPLC-ESI-MS/MS. The results revealed that 29 proteins exhibited significant changes after exposure. These proteins included surfactant-associated proteins (SP-A and SP-D), inflammatory proteins (complement component 3, immunoglobulins, lysozyme, etc.), growth factors (e.g., transforming growth factor alpha (TGF-alpha)), calcium-binding proteins (calcyclin, calgranulin A, calreticulin, and calvasculin), and other proteins (e.g., cathepsin D, saposin, and intestinal trefoil factor). To further evaluate changes in protein levels, a simple quantitative strategy was developed in this study. A large decrease in protein levels of SP-A and SP-D (0.24- and 0.38-fold, respectively) following exposure was observed. In contrast, protein levels of TGF-alpha and calcium-binding proteins were significantly increased (4.46- and 1.4-1.8-fold, respectively). Due to the diverse functions of these proteins, the results might contribute to understand the mechanisms involved in lung disorders induced by oil mist exposure.

Aerosols↗
PubMed1999

Proteomics for genetic and physiological studies in plants.

Proteomics is becoming a necessity in plant biology, as it is in medicine, zoology and microbiology, for deciphering the function and role of the genes that are or will be sequenced. In this review we focus on the various, mainly genetic, applications of the proteomic tools that have been developed in recent years: characterization of individuals or lines, estimation of genetic variability within and between populations, establishment of genetic distances that can be used in phylogenetic studies, characterization of mutants and localization of the genes encoding the revealed proteins. Improvements in specifically devoted software have permitted precise quantification of the variation in amounts of proteins, leading to the concept of "protein quantity loci" which, combined with the "quantitative trait loci" approach, results in testable hypotheses regarding the role of "candidate proteins" in the metabolism or phenotype under study. This new development is exemplified by the reaction of plants to drought, a trait of major agronomic interest. The accumulation of data regarding genomic and cDNA sequencing will be connected to the protein databases currently developed in plants.

Chromosome Mapping↗
PubMed2006

HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation.

Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples.

Chromatography, High Pressure Liquid↗
PubMed2004

Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.

Amino Acid Sequence↗
PubMed2004

Linear fuzzy gene network models obtained from microarray data by exhaustive search.

BACKGROUND: Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome. Gene network models are needed to interpret the resulting large and complex data sets. Rationally designed perturbations (e.g., gene knock-outs) can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis. We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling. RESULTS: We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle. Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance. Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes. CONCLUSION: Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation. Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system. Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems.

Artificial Intelligence↗
PubMed2003

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells.

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.

Amino Acid Sequence↗
PubMed2001

Desorption/ionization on silicon (DIOS): a diverse mass spectrometry platform for protein characterization.

Since the advent of matrix-assisted laser desorption/ionization and electrospray ionization, mass spectrometry has played an increasingly important role in protein functional characterization, identification, and structural analysis. Expanding this role, desorption/ionization on silicon (DIOS) is a new approach that allows for the analysis of proteins and related small molecules. Despite the absence of matrix, DIOS-MS yields little or no fragmentation and is relatively tolerant of moderate amounts of contaminants commonly found in biological samples. Here, functional assays were performed on an esterase, a glycosidase, a lipase, as well as exo- and endoproteases by using enzyme-specific substrates. Enzyme activity also was monitored in the presence of inhibitors, successfully demonstrating the ability of DIOS to be used as an inhibitor screen. Because DIOS is a matrix-free desorption technique, it also can be used as a platform for multiple analyses to be performed on the same protein. This unique advantage was demonstrated with acetylcholine esterase for qualitative and quantitative characterization and also by its subsequent identification directly from the DIOS platform.

Acetylcholine↗
PubMed2005

Mapping protein energy landscapes with amide hydrogen exchange and mass spectrometry: I. A generalized model for a two-state protein and comparison with experiment.

Protein amide hydrogen exchange (HDX) is a convoluted process, whose kinetics is determined by both dynamics of the protein and the intrinsic exchange rate of labile hydrogen atoms fully exposed to solvent. Both processes are influenced by a variety of intrinsic and extrinsic factors. A mathematical formalism initially developed to rationalize exchange kinetics of individual amide hydrogen atoms is now often used to interpret global exchange kinetics (e.g., as measured in HDX MS experiments). One particularly important advantage of HDX MS is direct visualization of various protein states by observing distinct protein ion populations with different levels of isotope labeling under conditions favoring correlated exchange (the so-called EX1 exchange mechanism). However, mildly denaturing conditions often lead to a situation where the overall HDX kinetics cannot be clearly classified as either EX1 or EX2. The goal of this work is to develop a framework for a generalized exchange model that takes into account multiple processes leading to amide hydrogen exchange, and does not require that the exchange proceed strictly via EX1 or EX2 kinetics. To achieve this goal, we use a probabilistic approach that assigns a transition probability and a residual protection to each equilibrium state of the protein. When applied to a small protein chymotrypsin inhibitor 2, the algorithm allows complex HDX patterns observed experimentally to be modeled with remarkably good fidelity. On the basis of the model we are now in a position to begin to extract quantitative dynamic information from convoluted exchange kinetics.

Algorithms↗
PubMed2005

Genomics and proteomics: role in the management of multiple sclerosis.

Epidemiological studies and neuro-imaging have provided important insights into the natural course and prognostic factors of multiple sclerosis (MS), but our ability to predict different courses of the disease, and especially its response to treatment, is still very limited. Pharmacogenetic, pharmacogenomic and proteomic studies aim to assess gene and protein function in disease and promise to help to fill this important gap in our knowledge. Such studies may increase our understanding of disease mechanisms and responses to therapeutic compounds. Large-scale transcriptional expression profiling can be performed using gene chip microarrays; this technology allows screening for differentially expressed genes without having well-defined underlying hypotheses ("discovery-driven research"). To complement the technique, real time reverse transcription and polymerase chain reaction (RT-PCR) can be used for more targeted profiling and provides quantitative data on pre-selected genes. However, to maximise their clinical utility, expression profiling results need to be combined with well-documented clinical and imaging data. Two forthcoming studies will investigate the long-term effects of early treatment with interferon beta-1b (IFNbeta) on the course of MS. The BENEFIT (BEtaseron/Betaferon in Newly Emerging MS for Initial Treatment) study will incorporate pharmacogenetic and pharmacogenomic analyses to determine the genetic elements controlling treatment response. BEST-PGx (Betaferon/Betaseron in Early relapsing-remitting MS Surveillance Trial-Pharmacogenomics) is an exploratory 2-year study that will investigate the value of RNA expression profiling and pharmacogenetics in predicting treatment response to IFNbeta in patients with early relapsing MS. The main goal of BEST-PGx is the identification of differences in gene expression profiles of patients showing differential treatment responses. In addition, this study may reveal new information relevant to the mechanism of action of interferon treatment in MS and also to differences in the underlying pathology of the immune system. These data may help us approach the goal of a really "individualised therapy" with increased efficacy, reduced adverse drug reactions and more efficient use of health care resources.

Adjuvants, Immunologic↗
PubMedDate not supplied

The role of mass spectrometry in plant systems biology.

Large-scale analyses of proteins and metabolites are intimately bound to advancements in MS technologies. The aim of these non-targeted "omic" technologies is to extend our understanding beyond the analysis of only parts of the system. Here, metabolomics and proteomics emerged in parallel with the development of novel mass analyzers and hyphenated techniques such as gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and multidimensional liquid chromatography coupled to mass spectrometry (LC-MS). The analysis of (i) proteins (ii) phosphoproteins, and (iii) metabolites is discussed in the context of plant physiology and environment and with a focus on novel method developments. Recently published studies measuring dynamic (quantitative) behavior at these levels are summarized; for these works, the completely sequenced plants Arabidopsis thaliana and Oryza sativa (rice) have been the primary models of choice. Particular emphasis is given to key physiological processes such as metabolism, development, stress, and defense. Moreover, attempts to combine spatial, tissue-specific resolution with systematic profiling are described. Finally, we summarize the initial steps to characterize the molecular plant phenotype as a corollary of environment and genotype.

Mass Spectrometry↗
PubMed2003

Hyperubiquitination of proteins in dilated cardiomyopathy.

Protein degradation by the ubiquitin-proteasome pathway plays an important role in a variety of fundamental cellular processes, including cell cycle regulation, transcription, antigen processing and muscle remodelling. Research into disorders associated with the ubiquitin-proteasome system has been mainly in the field of neurodegenerative diseases. It is however becoming increasingly apparent that defects in the system are responsible for a number of non-neurological pathologies. Based on initial observations made as part of a proteomic analysis of an animal model of dilated cardiomyopathy (DCM) which indicated increased activity of the ubiquitin-proteasome system, we sought to determine whether this system was perturbed in hearts of human DCM patients. We studied explanted hearts from 12 DCM, 9 ischaemic (IHD) and 12 unused donor hearts. Protein expression was examined using two-dimensional polyacrylamide gel electrophoresis, Western blotting and immunohistochemistry. Expression of mRNA was examined using real-time quantitative polymerase chain reaction. Ubiquitinated proteins were affinity purified using a ubiquitin-binding column and identified using peptide mass fingerprinting. All DCM hearts showed significantly higher expression of certain key enzymes of the ubiquitin-proteasome pathway. mRNA expression of ubiquitin carboxyl-terminal hydrolase (UCH) was significantly higher (5.4-fold) in DCM hearts than in control hearts. Myocytes in sections from DCM hearts stained positively for UCH, whereas control hearts were negative. Overall protein ubiquitination was increased two-fold in DCM relative to IHD hearts and five-fold relative to donor hearts. The ubiquitination of a number of distinct proteins was greatly enhanced in DCM hearts as revealed by anti-ubiquitin Western blots. A number of these proteins were identified using affinity purification and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Blotting, Western↗
PubMed2003

Natural selection of more designable folds: a mechanism for thermophilic adaptation.

An open question of great interest in biophysics is whether variations in structure cause protein folds to differ in the number of amino acid sequences that can fold to them stably, i.e., in their designability. Recently, we have shown that a novel quantitative measure of a fold's tertiary topology, called its contact trace, strongly correlates with the fold's designability. Here, we investigate the relationship between a fold's contact trace and its relative frequency of usage in mesophilic vs. thermophilic eubacteria. We observe that thermophilic organisms exhibit a bias toward using folds of higher contact trace when compared with mesophiles. We establish this difference both for the distributions of folds at the whole-proteome level and also through more focused structural comparisons of orthologous proteins. Our findings suggest that thermophilic adaptation in bacterial genomes occurs in part through natural selection of more designable folds, pointing to designability as a key component of protein fitness.

Adaptation, Physiological↗