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By admin, on May 16th, 2012
J Proteomics. 2012 May 8;
Parguiña AF, Rosa I, García A
Platelets play a fundamental role in haemostasis. Because they do not have a nucleus, proteomics is an ideal way to approach their biochemistry. Platelet proteomics is still a young field that emerged a decade ago. Initial platelet proteomics research focused on general proteome mapping followed by the exploration of sub-cellular compartments, the membrane proteome, and signaling pathways. The initial studies were later completed with the analysis of the platelet releasate and microparticles proteome. The success of these studies led to the application of platelet proteomics to the study of several pathologies where platelets play a fundamental role. Those include platelet-related disorders, such as storage pool disease, gray platelet syndrome, and Quebec platelet disorder; diseases where unwanted platelet activation is highly relevant, such as thrombosis and cardiovascular disease; and other diseases, such as cystic fibrosis, uremia, or Alzheimer's disease. In the present review article, we revise the most relevant proteomic studies on platelet-related diseases carried out to date, paying especial attention to sample preparation requirements for platelet clinical proteomic studies. This article is part of a Special Issue entitled: Integrated omics.
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Proteomics applied to the study of platelet-related diseases: Aiding the discovery of novel platelet biomarkers and drug targets.
By admin, on May 13th, 2012
Methods Mol Biol. 2012; 876: 67-82
Tang W
The plasma membrane (PM) controls cell's exchange of both material and information with the outside environment, and PM-associated proteins play key roles in cellular regulation. Numerous cell surface receptors allow cells to perceive and respond to various signals from neighbor cells, pathogens, or the environment; large numbers of transporter and channel proteins control material uptake or release. Quantitative proteomic analysis of PM-associated proteins can identify key proteins involved in signal transduction and cellular regulation. Here, we describe a protocol for quantitative proteomic analysis of PM proteins using two-dimensional difference gel electrophoresis. The protocol has been successfully employed to identify new components of the brassinosteroid signaling pathway, and should also be applicable to the studies of other plant signal transduction pathways and regulatory mechanisms.
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Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis.
By admin, on May 13th, 2012
Methods Mol Biol. 2012; 876: 67-82
Tang W
The plasma membrane (PM) controls cell's exchange of both material and information with the outside environment, and PM-associated proteins play key roles in cellular regulation. Numerous cell surface receptors allow cells to perceive and respond to various signals from neighbor cells, pathogens, or the environment; large numbers of transporter and channel proteins control material uptake or release. Quantitative proteomic analysis of PM-associated proteins can identify key proteins involved in signal transduction and cellular regulation. Here, we describe a protocol for quantitative proteomic analysis of PM proteins using two-dimensional difference gel electrophoresis. The protocol has been successfully employed to identify new components of the brassinosteroid signaling pathway, and should also be applicable to the studies of other plant signal transduction pathways and regulatory mechanisms.
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Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis.
By admin, on May 3rd, 2012
Antioxid Redox Signal. 2012 Apr 28;
Goosens V, Mars R, Akeroyd M, Vente A, Dreisbach A, Denham E, Kouwen T, van Rij T, Olsthoorn M, van Dijl JM
The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity. Here we have assessed whether proteomic techniques can be applied to probe the oxidative folding of membrane proteins using the Gram-positive bacterium Bacillus subtilis as a model organism. Specifically, we investigated the membrane proteome of a B. subtilis bdbCD mutant strain, which lacks the primary TDOR pair BdbC and BdbD, by gel-free mass spectrometry. In total, 18 membrane-associated proteins showed differing behavior in the bdbCD mutant and the parental strain. These included the ProA protein involved in osmoprotection. Consistent with the absence of ProA, the bdbCD mutant was found to be sensitive to osmotic shock. We conclude that membrane proteomics is a potentially effective approach to profile oxidative folding of bacterial membrane proteins.
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Is proteomics a reliable tool to probe the oxidative folding of bacterial membrane proteins?
By admin, on May 3rd, 2012
Antioxid Redox Signal. 2012 Apr 28;
Goosens V, Mars R, Akeroyd M, Vente A, Dreisbach A, Denham E, Kouwen T, van Rij T, Olsthoorn M, van Dijl JM
The oxidative folding of proteins involves disulfide bond formation, which is usually catalyzed by thiol-disulfide oxidoreductases (TDORs). In bacteria, this process takes place in the cytoplasmic membrane and other extracytoplasmic compartments. While it is relatively easy to study oxidative folding of water-soluble proteins on a proteome-wide scale, this has remained a major challenge for membrane proteins due to their high hydrophobicity. Here we have assessed whether proteomic techniques can be applied to probe the oxidative folding of membrane proteins using the Gram-positive bacterium Bacillus subtilis as a model organism. Specifically, we investigated the membrane proteome of a B. subtilis bdbCD mutant strain, which lacks the primary TDOR pair BdbC and BdbD, by gel-free mass spectrometry. In total, 18 membrane-associated proteins showed differing behavior in the bdbCD mutant and the parental strain. These included the ProA protein involved in osmoprotection. Consistent with the absence of ProA, the bdbCD mutant was found to be sensitive to osmotic shock. We conclude that membrane proteomics is a potentially effective approach to profile oxidative folding of bacterial membrane proteins.
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Is proteomics a reliable tool to probe the oxidative folding of bacterial membrane proteins?
By admin, on May 1st, 2012
Drug Test Anal. 2012 Apr 29;
Nikolovski Z, De La Torre C, Chiva C, Borràs E, Andreu D, Ventura R, Segura J
Mature red blood cells (RBCs) are the end-stage of a development process that starts in the bone marrow and continues to differentiate, through reticulocyte stage, entering into the circulation with a four-month lifespan. While stored, RBCs undergo different changes. The aim of this study was to evaluate changes occurring in RBC membranes during storage that could be used as possible markers to detect the misuse of blood transfusion in sports. Whole blood was collected from two volunteers in blood bags and stored for 42 days at 4°C. At different times (1, 7, 21, and 42 days of storage) whole blood was extracted under sterile conditions and submitted to RBC membrane ghost preparation and further analysis. Proteomic methods were applied using two strategies: protein oriented using 2-DE gels and peptide oriented using isobaric tags for relative and absolute quantitation (iTRAQ). In both approaches, the goal was to compare detectable changes in RBC membrane proteome before and after standard storage at different times. Some of the changes were confirmed with both methodologies employed, while with others only with one of them. Complementarities of the methods in this case showed to be an advantage. Changes were observed in two different protein complexes. In one of them, changes consisted of proteins decreasing, while increasing in the other during storage of RBCs. They are mostly located in cytoskeleton – spectrin β, band 4.2, ankyrin-1, tropomodulin-1, β adducin, band 4.9 (dematin), tropomyosin, while some changes were also observed in transmembrane proteins (glycophorin C, aquaporin-1, band 3). Copyright © 2012 John Wiley & Sons, Ltd.
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Alterations of the erythrocyte membrane proteome and cytoskeleton network during storage – a possible tool to identify autologous blood transfusion.
By admin, on April 29th, 2012
J Proteomics. 2012 Apr 17;
Alexandre BM, Charro N, Blonder J, Lopes C, Azevedo P, de Almeida AB, Chan KC, Prieto DA, Issaq H, Veenstra TD, Penque D
Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: “Integrated omics – Functional applications to blood and blood therapeutics”.
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Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.
By admin, on April 29th, 2012
J Proteomics. 2012 Apr 17;
Alexandre BM, Charro N, Blonder J, Lopes C, Azevedo P, de Almeida AB, Chan KC, Prieto DA, Issaq H, Veenstra TD, Penque D
Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: “Integrated omics – Functional applications to blood and blood therapeutics”.
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Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.
By admin, on April 28th, 2012
Proteomics. 2012 Mar; 12(6): 845-58
Gesslbauer B, Poljak A, Handwerker C, Schüler W, Schwendenwein D, Weber C, Lundberg U, Meinke A, Kungl AJ
The versatility of the surface of Borrelia, the causative agent of Lyme borreliosis, is very important in host-pathogen interactions allowing bacteria to survive in ticks and to persist in a mammalian environment. To identify the surface proteome of Borrelia, we have performed a large comparative proteomic analysis on the three most important pathogenic Borrelia species, namely B. burgdorferi (strain B31), B. afzelii (strain K78), and B. garinii (strain PBi). Isolation of membrane proteins was performed by using three different approaches: (i) a detergent-based fractionation of outer membrane proteins; (ii) a trypsin-based partial shedding of outer cell surface proteins; (iii) biotinylation of membrane proteins and preparation of the biotin-labelled fraction using streptavidin. Proteins derived from the detergent-based fractionation were further sub-fractionated by heparin affinity chromatography since heparin-like molecules play an important role for microbial entry into human cells. All isolated proteins were analysed using either a gel-based liquid chromatography (LC)-MS/MS technique or by two-dimensional (2D)-LC-MS/MS resulting in the identification of 286 unique proteins. Ninety seven of these were found in all three Borrelia species, representing potential targets for a broad coverage vaccine for the prevention of Lyme borreliosis caused by the different Borrelia species.
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Comparative membrane proteome analysis of three Borrelia species.
By admin, on April 8th, 2012
This paper investigated the outer membrane proteins (OMPs) of E. coli K-12 by proteomic approach, especially functional proteomics. On the … Subject, Escherichia coli K-12, outer membrane protein, Proteomics,. FileType …
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Proteomic Analysis on Outer Membrane Proteins of Escherichia Coli …
By admin, on April 8th, 2012
This paper investigated the outer membrane proteins (OMPs) of E. coli K-12 by proteomic approach, especially functional proteomics. On the … Subject, Escherichia coli K-12, outer membrane protein, Proteomics,. FileType …
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Proteomic Analysis on Outer Membrane Proteins of Escherichia Coli …
By admin, on March 28th, 2012
J Proteomics. 2012 Mar 17;
Haußmann U, Poetsch A
Corynebacterium glutamicum can utilize various monocyclic aromatic carbon sources, including protocatechuate, which is catabolized via the β-ketoadipate pathway. In order to obtain a global survey of occurring physiological adaptations on the proteome level, cytoplasmic and membrane fraction from cells grown on protocatechuate or glucose as sole carbon and energy source were compared. Shotgun proteomics and relative protein quantification with metabolic isotope labeling and spectral counting were employed. Altogether, 139 proteins were found to change their abundance during growth on protocatechuate. A general adaptation of energy metabolism to meet increased energy production by oxidative phosphorylation and a stress response occurred. Adjustments of carbon and amino acid metabolism in the cytoplasmic and membrane proteome were indicative of a starvation response. The different regulation of porins and cell wall biosynthesis proteins suggests a change in its architecture upon assimilation of the aromatic carbon source. Some of the observed changes could be explained by an involvement of the GlxR and McbR regulons.
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Global proteome survey of protocatechuate- and glucose-grown Corynebacterium glutamicum reveals multiple physiological differences.
By admin, on March 28th, 2012
J Proteomics. 2012 Mar 17;
Haußmann U, Poetsch A
Corynebacterium glutamicum can utilize various monocyclic aromatic carbon sources, including protocatechuate, which is catabolized via the β-ketoadipate pathway. In order to obtain a global survey of occurring physiological adaptations on the proteome level, cytoplasmic and membrane fraction from cells grown on protocatechuate or glucose as sole carbon and energy source were compared. Shotgun proteomics and relative protein quantification with metabolic isotope labeling and spectral counting were employed. Altogether, 139 proteins were found to change their abundance during growth on protocatechuate. A general adaptation of energy metabolism to meet increased energy production by oxidative phosphorylation and a stress response occurred. Adjustments of carbon and amino acid metabolism in the cytoplasmic and membrane proteome were indicative of a starvation response. The different regulation of porins and cell wall biosynthesis proteins suggests a change in its architecture upon assimilation of the aromatic carbon source. Some of the observed changes could be explained by an involvement of the GlxR and McbR regulons.
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Global proteome survey of protocatechuate- and glucose-grown Corynebacterium glutamicum reveals multiple physiological differences.
By admin, on March 27th, 2012
Aquat Toxicol. 2012 Mar 3; 114-115C: 189-199
Huang X, Huang HQ
Methyl parathion (MP) is a widely used organophosphorus pesticide that causes severe health and environmental effects. We investigated the alteration of the proteomic profile in the membrane enriched fraction of the kidneys of the scallop Mizuhopecten yessoensis exposed to low-level MP. Gas chromatography analysis showed that MP residues were significantly accumulated in the kidneys and the digestive glands of the scallops. According to two-dimensional electrophoresis, 17 proteins were differentially modulated under MP exposure. The mRNA expressions of 12 differential proteins were analyzed using quantitative PCR, and 10 showed consistent alteration of mRNA level with that of protein expression level. Altered expressions of two proteins (mitochondrial processing peptidase and α-tubulin) were also examined using Western blotting, showing that the mitochondrial processing peptidase was down-regulated but α-tubulin remained unchanged in response to MP exposure. Subcellular locations of all the identified proteins that were predicted using bioinformatics tools indicate that few of them are permanently located in the membrane. The differentially expressed proteins are involved in several critical biological processes, and their relevance to human health has been illuminated. These data taken together have provided some novel insights into the chronic toxicity mechanism of MP and have suggested mitochondrial processing peptidase as a potential biomarker for human health and environmental monitoring.
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Alteration of the kidney membrane proteome of Mizuhopecten yessoensis induced by low-level methyl parathion exposure.
By admin, on March 27th, 2012
Aquat Toxicol. 2012 Mar 3; 114-115C: 189-199
Huang X, Huang HQ
Methyl parathion (MP) is a widely used organophosphorus pesticide that causes severe health and environmental effects. We investigated the alteration of the proteomic profile in the membrane enriched fraction of the kidneys of the scallop Mizuhopecten yessoensis exposed to low-level MP. Gas chromatography analysis showed that MP residues were significantly accumulated in the kidneys and the digestive glands of the scallops. According to two-dimensional electrophoresis, 17 proteins were differentially modulated under MP exposure. The mRNA expressions of 12 differential proteins were analyzed using quantitative PCR, and 10 showed consistent alteration of mRNA level with that of protein expression level. Altered expressions of two proteins (mitochondrial processing peptidase and α-tubulin) were also examined using Western blotting, showing that the mitochondrial processing peptidase was down-regulated but α-tubulin remained unchanged in response to MP exposure. Subcellular locations of all the identified proteins that were predicted using bioinformatics tools indicate that few of them are permanently located in the membrane. The differentially expressed proteins are involved in several critical biological processes, and their relevance to human health has been illuminated. These data taken together have provided some novel insights into the chronic toxicity mechanism of MP and have suggested mitochondrial processing peptidase as a potential biomarker for human health and environmental monitoring.
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Alteration of the kidney membrane proteome of Mizuhopecten yessoensis induced by low-level methyl parathion exposure.
By admin, on March 13th, 2012
J Proteomics. 2012 Mar 3;
Meisrimler CN, Lüthje S
The crucial cellular role of membrane proteins is generally known for all life forms. Depending on the species, tissue, compartment, functions and physiological conditions, membranes differ in their protein and lipid profiles. Additionally, occurrence of microdomains hampers quantitative protein solubilisation and therefore membrane proteomics remain a major challenge. In the present study sample preparation (TCA-acetone and methanol-chloroform precipitation with and without SDS pre-solubilisation) for two-dimensional PAGE were compared for microsomal fractions of leaves (Arabidopsis thaliana, Nicotiana tabaccum, Pisum sativum) and roots (P. sativum, Zea mays). Generally, pre-solubilisation with SDS impaired the resolution of the gels. All samples showed higher spot yields with TCA-acetone precipitation. Finally, we compared the results of conventional 2D-PAGE (IPG/SDS-PAGE) and the combination of off-gel fractionation in the first-dimension, 10% urea-SDS-PAGE in the second-dimension. Results showed that more spots are present in the alkaline pH range after off-gel fractionation then on conventional 2D-PAGE. For the first time, off-gel fractionation was combined with SDS/SDS-PAGE and BAC/SDS-PAGE to improve the resolution after off-gel fractionation. Transmembrane domains and GRAVY were calculated for all significant identified spots resulting from the MALDI-TOF-TOF mass spectrometry showing that in the second dimension after off-gel fractionation 10.3% more transmembrane proteins were identified compared to IPG/SDS-PAGE.
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IPG-strips versus off-gel fractionation: Advantages and limits of two-dimensional PAGE in separation of microsomal fractions of frequently used plant species and tissues.
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