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PubMed2005

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.

Amino Acid Sequence↗
PubMed2005

Nuclear and mitochondrial DNA repair: similar pathways?

Mitochondrial DNA (mtDNA) alterations are implicated in a broad range of human diseases and alterations of the mitochondrial genome are assumed to be a result of its high susceptibility to oxidative damage and its limited DNA repair compared to nuclear DNA (nDNA). Characterization of DNA repair mechanisms has generally focused on these processes in nDNA but increasing interest and research effort have contributed to our knowledge of the mechanisms underlying DNA repair in mitochondria. In this review, we make comparisons between nDNA and mtDNA repair pathways and propose a model for how these pathways interact in mitochondria.

Animals↗
PubMed2003

Comparing protein abundance and mRNA expression levels on a genomic scale.

Attempts to correlate protein abundance with mRNA expression levels have had variable success. We review the results of these comparisons, focusing on yeast. In the process, we survey experimental techniques for determining protein abundance, principally two-dimensional gel electrophoresis and mass-spectrometry. We also merge many of the available yeast protein-abundance datasets, using the resulting larger 'meta-dataset' to find correlations between protein and mRNA expression, both globally and within smaller categories.

Animals↗
PubMed2026

Machine Learning-Driven Prediction of Coronary Artery Disease Risk Based on UK Biobank Plasma Proteomics.

BACKGROUND: Coronary artery disease (CAD) is a leading global cause of mortality, yet the predictive accuracy of conventional risk models is limited. Here, we integrate conventional risk factors, polygenic risk scores, and large-scale proteomics to develop a unified model for enhanced CAD risk prediction. METHODS: Using data from UK Biobank, participants with plasma proteomics and genetic risk data were included after excluding prevalent CAD. Participants from England were split into training (n=32 330) and internal validation (n=13 857) sets, and Scotland/Wales participants formed an external validation set (n=5775). Incident CAD was ascertained from linked health records. A 202-protein proteomic risk score was derived by least absolute shrinkage and selection operator Cox regression, and CatBoost models were trained using conventional risk factors alone and with incremental addition of polygenic risk scores and protein proteomic risk scores; Shapley Additive Explanations-guided forward selection identified a compact protein panel. RESULTS: Across cohorts, the median age was 58 years and ∼45% were men. Protein proteomic risk score was dose-dependently associated with CAD risk. Compared with conventional risk factors alone, integrating polygenic risk scores and protein proteomic risk scores improved discrimination, with the area under the curve increasing from 0.750 (95% CI, 0.732-0.767) to 0.789 (95% CI, 0.772-0.805) in internal validation and from 0.717 (95% CI, 0.683-0.750) to 0.762 (95% CI, 0.732-0.791) in external validation. A 9-protein panel (GDF15 [growth differentiation factor 15], MMP12 [matrix metalloproteinase 12], NPPB [natriuretic peptide B], PGF [placental growth factor], REN [renin], ADGRG2 [adhesion G-protein coupled receptor], ACE2 [angiotensin-converting enzyme 2], CDCP1 [CUB domain-containing protein 1], CXCL17 [C-X-C motif chemokine ligand 17)]) captured most proteomic predictive information. CONCLUSIONS: Our findings demonstrate that integrating conventional risk factors, polygenic risk scores, and proteomic data improves CAD risk prediction. This study highlights the utility of proteomics in precision cardiovascular medicine and simplified risk stratification tools.

Humans↗
PubMed2006

Evolutionary and structural feedback on selection of sequences for comparative analysis of proteins.

It has been noted that slowly evolving protein residues have two properties: (a) they tend to cluster in the native fold, and (b) they delineate functional surfaces-parts of the surface through which the protein interacts with other proteins or small ligands. Herein, we demonstrate that the two are coupled sufficiently strongly that one effect, when observed, statistically implies the other. Detection of both can be accomplished in multiple sequence alignment related methods by the careful selection of relevant sequences. For the demonstration, we use two sets of protein families: a small set of diverse proteins with diverse functional surfaces, and a large set of homodimerizing enzymes. A practical outcome of our considerations is a simple prescriptive rule for the selection of homologous sequences for the comparative analysis of proteins: in order to optimize the detection of (potentially unknown) functional surfaces, it is sufficient to select sequences in such a way that the residues observed at any level of evolutionary divergence, as implied by the alignment, cluster on the folded protein.

Animals↗
PubMed2006

Modeling cellular machinery through biological network comparison.

Molecular networks represent the backbone of molecular activity within the cell. Recent studies have taken a comparative approach toward interpreting these networks, contrasting networks of different species and molecular types, and under varying conditions. In this review, we survey the field of comparative biological network analysis and describe its applications to elucidate cellular machinery and to predict protein function and interaction. We highlight the open problems in the field as well as propose some initial mathematical formulations for addressing them. Many of the methodological and conceptual advances that were important for sequence comparison will likely also be important at the network level, including improved search algorithms, techniques for multiple alignment, evolutionary models for similarity scoring and better integration with public databases.

Algorithms↗
PubMed2000

Relationship between whole proteome aminoacid composition and static DNA curvature.

To study possible relationships between an organism's genomic DNA curvature and the aminoacid composition of its proteome, every peptidic sequence from fully determined genomes was retrotranslated using the E. coli codon preferences, and the curvature profiles of the resulting DNA sequences were calculated and compared. A clear interdependence between these two variables was observed, as each retrotranslated proteome presented a distinctive, statistically significant DNA curvature profile biased toward its natural DNA curvature profile. In addition, by comparing the profiles arising from real and randomly permuted proteomes, we also found a position-dependent contribution of the peptidic sequence to DNA curvature. The implications of these results support the idea of a possible selection toward a specific global curvature of genomes.

Amino Acids↗
PubMed2004

Functional specialization amongst the Arabidopsis Toc159 family of chloroplast protein import receptors.

The initial stages of preprotein import into chloroplasts are mediated by the receptor GTPase Toc159. In Arabidopsis thaliana, Toc159 is encoded by a small gene family: atTOC159, atTOC132, atTOC120, and atTOC90. Phylogenetic analysis suggested that at least two distinct Toc159 subtypes, characterized by atToc159 and atToc132/atToc120, exist in plants. atTOC159 was strongly expressed in young, photosynthetic tissues, whereas atTOC132 and atTOC120 were expressed at a uniformly low level and so were relatively prominent in nonphotosynthetic tissues. Based on the albino phenotype of its knockout mutant, atToc159 was previously proposed to be a receptor with specificity for photosynthetic preproteins. To elucidate the roles of the other isoforms, we characterized Arabidopsis knockout mutants for each one. None of the single mutants had strong visible phenotypes, but toc132 toc120 double homozygotes appeared similar to toc159, indicating redundancy between atToc132 and atToc120. Transgenic complementation studies confirmed this redundancy but revealed little functional overlap between atToc132/atToc120 and atToc159 or atToc90. Unlike toc159, toc132 toc120 caused structural abnormalities in root plastids. Furthermore, when proteomics and transcriptomics were used to compare toc132 with ppi1 (a receptor mutant that is specifically defective in the expression, import, and accumulation of photosynthetic proteins), major differences were observed, suggesting that atToc132 (and atToc120) has specificity for nonphotosynthetic proteins. When both atToc159 and the major isoform of the other subtype, atToc132, were absent, an embryo-lethal phenotype resulted, demonstrating the essential role of Toc159 in the import mechanism.

Arabidopsis↗
PubMed2005

Probabilistic model of the human protein-protein interaction network.

A catalog of all human protein-protein interactions would provide scientists with a framework to study protein deregulation in complex diseases such as cancer. Here we demonstrate that a probabilistic analysis integrating model organism interactome data, protein domain data, genome-wide gene expression data and functional annotation data predicts nearly 40,000 protein-protein interactions in humans-a result comparable to those obtained with experimental and computational approaches in model organisms. We validated the accuracy of the predictive model on an independent test set of known interactions and also experimentally confirmed two predicted interactions relevant to human cancer, implicating uncharacterized proteins into definitive pathways. We also applied the human interactome network to cancer genomics data and identified several interaction subnetworks activated in cancer. This integrative analysis provides a comprehensive framework for exploring the human protein interaction network.

Chromosome Mapping↗
PubMed2004

The Schistosoma mansoni soluble proteome: a comparison across four life-cycle stages.

Differential analysis of immune responses to schistosomes has routinely been performed using complex mixtures of soluble proteins from various life-cycle stages, on the assumption that these differed significantly in composition. Proteomic techniques now allow us to characterise and compare such mixtures. The soluble proteins from cercariae, lung-schistosomula, adult worms and eggs of Schistosoma mansoni were separated by high-resolution two-dimensional electrophoresis and the resulting images analysed using appropriate software. A high degree of quantitative and qualitative similarity in spot pattern was revealed across the life-cycle, greatest between adjacent stages. To initiate mapping of these soluble proteomes, the 40 most abundant spots in each preparation, accounting for 21-46% of the total protein, were subjected to peptide fingerprinting by mass spectrometry. On average 55% of the spots were identified, but overall, these comprised only 32 different protein species. With one exception all proteins originated in the cytosol and 24 of the 32 had previously been pinpointed by virtue of their immunoreactivity, including four of the WHO priority vaccine candidates. The similarity in composition between the four preparations means that they are unlikely to discriminate adequately between immune responses to different life-cycle stages and argues strongly for the need to identify true stage-specific marker proteins. Equally, it is difficult to reconcile the abundance and immunogenicity of such cytosolic proteins with their status as vaccine candidates, as it is unlikely they will be accessible to the immune system in an intact parasite.

Animals↗
PubMed2001

Chemiluminescent standards for quantitative comparison of two-dimensional electrophoresis western blots.

Chemiluminescent probes offer highly sensitive quantitative analyses of proteins blotted from electrophoretic gels onto a supporting matrix (e.g. nitrocellulose or polyvinylidene difluoride). Visualization of signals from probes involves the emission of light that is dependent on a number of variables (e.g. conjugated enzyme activity, antibody titer, hybridization efficiency, substrate concentration, chemical reactivity, temperature, etc.). Thus, it is important to be able to correct for these variations. For example, the exposure time of the blot to the detection medium (e.g., film or digital camera) is a critical variable in the final results. Several protein samples separated on a single blot (e.g. one-dimensional resolution) can be compared from the ratio of the individual proteins, but comparison of separate blots completed on different days requires a chemiluminescent standard. The situation is more complex when only one sample per gel/blot is used (i.e. two-dimensional electrophoresis (2-DE)). This paper describes a method for preparing agarose embedded standardized strips that contain dilutions of antigens that can be visualized with the corresponding probe antibody. This standardization strip can be produced from common laboratory supplies and provides a method to correct for alterations in chemiluminescent intensities from different 2-DE analysis. Several standardization strips can be produced simultaneously and then used during the electroblotting step of different blots on different days.

Animals↗
PubMed2007

2-D DIGE analysis of liver and red blood cells provides further evidence for oxidative stress in schizophrenia.

The molecular disease mechanisms associated with schizophrenia remain largely unknown. Although primarily considered a disorder of the brain, there is evidence of a peripheral component to schizophrenia. In this study, we investigated liver tissue and red blood cells (RBC) from schizophrenia patients and controls using 2-D DIGE proteomic analysis. Fourteen proteins were significantly altered in liver samples from schizophrenia patients (n = 15) compared to healthy controls (n = 15). Analysis of the schizophrenia RBC proteome revealed 8 proteins significantly altered in samples from schizophrenia patients (13 antipsychotic-treated and 7 drug-naïve) compared to controls (n = 20). Six of the altered proteins in the liver and four of the altered RBC proteins are related to oxidative stress. These results corroborate our earlier findings obtained from post-mortem brain studies and substantiate our hypothesis that metabolic alterations leading to oxidative stress are linked to the schizophrenia disease process. Our results also suggest that at least some of the pathological processes associated with the schizophrenia disease process can be traced in peripheral tissue. If peripheral cells can be used as a disease surrogate, promising new investigative avenues could be explored.

Adult↗
PubMed2005

Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp.

Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron-sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.

Amino Acid Sequence↗
PubMed2005

Identification of leukemia-associated antigens in chronic myeloid leukemia by proteomic analysis.

The immune system plays an important role in the treatment of chronic myeloid leukemia (CML). Identification of leukemia-associated antigens (LAAs) eliciting an immune response in patients is a prerequisite for specific immunotherapy of CML. To identify new LAAs in CML, We utilized a novel approach based serology and proteomics technologies. LAAs were identified by comparing the reactivity of proteins resolved by 2-DE with sera from CML patients and healthy donors. Several new LAAs were identified including alpha enolase, aldolase A, HSP70 protein8, beta-tubulin and tropomyosin isoforms. Although, the functions of these identified proteins in CML need further investigation, the detection of autoantibodies in CML may have value on CML screening, diagnosis, or follow-up. Additionally, identification of LAAs in CML may also be of vital importance in antigen-based immunotherapy.

Adolescent↗
PubMed2004

Bone protects proteins over thousands of years: extraction, analysis, and interpretation of extracellular matrix proteins in archeological skeletal remains.

In a good state of preservation, bone conserves the entire protein pattern of extracellular bone matrix proteins over thousands of years. The quality of the profiles of matrix proteins isolated from ancient bones (ranging from the pre-Pottery Neolithic Phase to Early Modern Times from different archaeological sites in different geographical areas), separated by electrophoresis, is as good as those from recent bones. Molecules arising from collagenous proteins (e.g., collagen type I), from the noncollagenous group (e.g., osteonectin), and from the immune system (e.g., immunoglobulin G) were identified in Western blots by specific antibodies. A comparative study of the immunoglobulin G content of the bones of five prehistoric children showed the lowest immunoglobulin G content in a child who suffered from chronic scurvy. Ancient bone proteins were also separated by two-dimensional electrophoresis. This technique makes fractionation of the complex protein mixtures of extracellular bone matrix more reproducible. Bone retains a chemical memory of earlier metabolic stimuli in its configuration of collagenous and noncollagenous proteins. In combination with the results of the microscopic examination of ancient bone, it should be possible to obtain more reliable information on the history and the evolution of diseases, based on analysis of intact proteins.

Adolescent↗
PubMed2005

Mouse anterior pituitary gland: analysis by ion trap mass spectrometry.

We investigated the proteome of the anterior pituitary gland (AP) in a species in which the genome has been sequenced. Subcellular fractions of APs from 2-month-old male mice were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography tandem mass spectrometry and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 g and cytosolic fractions, we identified 49, 36 and 68 different proteins, respectively. A total of 115 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the alpha-subunit for the glycoprotein hormones, and luteinizing hormone-beta. Groups of other identified proteins included hormone-processing, secretion granule-associated, non-hormonal endoplasmic reticulum-associated, calcium-binding, protein kinase C-associated, histones, non-histone chromosomal, other RNA-binding, heterogeneous nuclear ribonucleoproteins, splicing factors, helicases, lamins, ribosomal, microtubule-associated, microfilament-associated, adenosine triphosphate- and guanosine triphosphate-associated, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation, enzymes in glycolysis and the tricarboxylic and urea cycles and the pentose phosphate path, heat-shock, glutathione-associated, peroxidases, ubiquitin-associated, catabolic, protease inhibitors, other, and blood proteins. The 115 proteins reported in this study and the 145 proteins reported in a previous study on the AP of the adult male Golden Syrian hamster are compared and form a foundation for defining the proteome in normal adult male AP.

Animals↗
PubMed2005

Identification of differentially expressed genes between fetal and adult mouse kidney: candidate gene in kidney development.

BACKGROUND: The kidney development involves a wide variety of developmental processes requiring a lot of genes expressed in a sequential manner. The aim of the present study is to identify new genes involved in these processes. METHODS: To obtain a view of the mouse embryonic kidney transcriptome we used the SADE method, which allows large-scale quantitative gene expression measurements. RESULTS: 7,689 tags were sequenced from our library. Among the 4,507 unique transcripts yielded, 64% correspond to known genes, 22% ESTs, 12% unidentified genes. 472 genes were differentially expressed as compared to published adult kidney library. Among these, we identified several candidate genes and focused on a particular one: thymosin beta4 (Tbeta4), an actin-sequestering protein more highly expressed in fetal kidney. First we studied the in vivo expression patterns of Tbeta4 transcript during kidney development. Tbeta4 increases throughout the kidney development and remains high during active nephrogenesis. Moreover, the spatial distribution of Tbeta4 mRNA was analysed and reveals that during active nephrogenesis (i.e., 18 dpc) Tbeta4 is localised in differentiating glomeruli. In adult kidney, Tbeta4 remains expressed in podocytes and collecting ducts. CONCLUSION: Our results provide the first demonstration of Tbeta4 production in vivo by embryonic kidney and further show that Tbeta4 is implicated in kidney organogenesis.

Aging↗
PubMed2005

Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling.

Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status.

Animals↗