Home About us Contact
|
Home About us Contact | ||
|
|
||
Membrane Proteome (membrane + proteome)
Selected AbstractsA novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DEELECTROPHORESIS, Issue 23 2009Gloria Alvarez-Llamas Abstract With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, we propose here a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. We investigate here different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE, paying particular attention to hemoglobin removal. A simple, quick and satisfactory approach for hemoglobin depletion based on HemogloBindÔ reagent was satisfactorily applied to erythrocyte cells, allowing the analysis of the cytosolic sub-proteome by 2-DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described here, allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and well-resolved 2-DE patterns. An aliquot of the membrane fraction was also in solution digested and analyzed by nano-LC coupled to an LTQ-Orbitrap mass spectrometer. A total of 188 unique proteins were identified by this approach. This study sets the basis for future clinical studies where the erythrocyte cell may be implicated. [source] Analysis of integral membrane proteins by heat gel-embedment combined with improved in-gel digestionsELECTROPHORESIS, Issue 23 2009Jian Zhou Abstract Analysis of integral membrane proteins (IMPs) presents a special challenge because of their hydrophobic nature and low abundance. Here, a new method was developed, which involved heat gel-embedment and improved in-gel digestion of the proteins. Membrane protein lysate containing detergents was mixed with acrylamide solution and the proteins were embedded when the gel polymerized. For comparison, the protein embedment was made at different temperatures (25, 35 or 45°C), and the in-gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS), 0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. Compared with that at 25°C, gel-embedment at 45°C improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%. 0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel-embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel-embedment combined with improved in-gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of integral membrane proteome. [source] Defining the membrane proteome of NK cellsJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2010Dhimankrishna Ghosh Abstract The present study was initiated to define the composition of the membrane proteome of the Natural Killer (NK) like cell line YTS. Isolated membranes were treated with reagents that have been reported to remove peripheral membrane proteins. Additional steps involving trifluoroethanol (TFE) were introduced in an effort to remove remaining nonintegral membrane proteins. This treatment resulted in the release of a subset of proteins without any apparent disruption of membrane integrity. The membranes were solubilized and digested with trypsin in 25% TFE. The resulting peptides were separated using an off-line two-dimensional reversed phase LC technique at alkaline and acidic pHs. Mass spectrometric analysis identified 1843 proteins with high confidence scores. On the basis of the presence of transmembrane regions or evidence of posttranslational modifications and prediction algorithms, approximately 40% of the identified proteins were predicted as plausible membrane proteins. The remaining species were largely involved in cellular processes and molecular functions that could be predicted to be transiently associated with membranes. The analytical approaches presented in this study offer robust generic methods for the identification and characterization of membrane proteins. These observations highlight the fact that the membrane is a dynamic entity that is composed of integral and stably associated proteins. Copyright © 2009 John Wiley & Sons, Ltd. [source] Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source , a membrane proteome-centric viewPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2009Ute Haußmann Abstract The ability of microorganisms to assimilate aromatic substances as alternative carbon sources is the basis of biodegradation of natural as well as industrial aromatic compounds. In this study, Corynebacterium glutamicum was grown on benzoate as sole carbon and energy source. To extend the scarce knowledge about physiological adaptation processes occurring in this cell compartment, the membrane proteome was investigated under quantitative and qualitative aspects by applying shotgun proteomics to reach a comprehensive survey. Membrane proteins were relatively quantified using an internal standard metabolically labeled with 15N. Altogether, 40 proteins were found to change their abundance during growth on benzoate in comparison to glucose. A global adaptation was observed in the membrane of benzoate-grown cells, characterized by increased abundance of proteins of the respiratory chain, by a starvation response, and by changes in sulfur metabolism involving the regulator McbR. Additional to the relative quantification, stable isotope-labeled synthetic peptides were used for the absolute quantification of the two benzoate transporters of C. glutamicum, BenK and BenE. It was found that both transporters were expressed during growth on benzoate, suggesting that both contribute substantially to benzoate uptake. [source] Analysis of the membrane proteome of canine pancreatic rough microsomes identifies a novel Hsp40, termed ERj7PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2009René P. Zahedi Abstract The rough ER (rER) plays a central role in the biogenesis of most extracellular and many organellar proteins in eukaryotic cells. Cells that are specialized in protein secretion, such as pancreatic cells, are particularly rich in rER. In the process of cell homogenization, the rER is converted into ribosome-studded vesicles, the so-called rough microsomes. Here we report on a membrane proteomic analysis of canine pancreatic rough microsomes. Special emphasis was placed on components involved in the various aspects of protein biogenesis, such as protein transport, protein folding, protein modification, and protein degradation. Our results indicate that the Hsp70-chaperone network that is present in the pancreatic ER is even more complex than previously thought, and suggest that the pancreatic rER has a significant capacity for protein degradation. [source] Outer membrane proteome of Actinobacillus pleuropneumoniae: LC-MS/MS analyses validate in silico predictionsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2007Jacqueline W. Chung Abstract The Gram-negative bacterial pathogen Actinobacillus pleuropneumoniae causes porcine pneumonia, a highly infectious respiratory disease that contributes to major economic losses in the swine industry. Outer membrane (OM) proteins play key roles in infection and may be targets for drug and vaccine research. Exploiting the genome sequence of A. pleuropneumoniae serotype 5b, we scanned in silico for proteins predicted to be localized at the cell surface. Five genome scanning programs (Proteome Analyst, PSORT-b, BOMP, Lipo, and LipoP) were run to construct a consensus prediction list of 93 OM proteins in A. pleuropneumoniae. An inventory of predicted OM proteins was complemented by proteomic analyses utilizing gel- and solution-based methods, both coupled to LC-MS/MS. Different protocols were explored to enrich for OM proteins; the most rewarding required sucrose gradient centrifugation followed by membrane washes with sodium bromide and sodium carbonate. This protocol facilitated our identification of 47 OM proteins that represent 50% of the predicted OM proteome, most of which have not been characterized. Our study establishes the first OM proteome of A. pleuropneumoniae. [source] Insights into the membrane proteome of rat liver peroxisomes: Microsomal glutathione-S-transferase is shared by both subcellular compartmentsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Markus Islinger Dr. Abstract Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl- n -hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation. [source] Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracyclinePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2006Changxin Xu Abstract The elucidation of the molecular details of antibiotic resistance will lead to improvements in extending the efficacy of current antimicrobials. In the current study, proteomic methodologies were applied to characterize functional outer membrane proteins (Omps) of E.,coli,K-12 responded to tetracycline and ampicillin resistance for understanding of universal pathways that form barriers for antimicrobial agents. For this purpose, E.,coli,K-12 expressional outer membrane proteome was characterized and identified with the use of 2-DE and MALDI-TOF/MS methods. Then, differential Omps due to tetracycline or ampcilin resistance were determined by comparison between tetracycline minimum inhibitory concentration (MIC)10, ampicillin,MIC10, control0 and control10, showing 9,proteins with 11,spots for tetracycline and 8,protein with 9,spots for ampicillin, showing a difference in only 1,protein (decreased LamB in tetracyclin) between the two antibiotics. Among the proteins, 3,were known as antibiotic-resistant proteins, including TolC, OmpC and YhiU, while FimD precursor, LamB, Tsx, YfiO, OmpW, NlpB were first reported here to be antibiotic-resistance-related proteins. Our findings will be helpful for further understanding of antibiotic-resistant mechanism(s). This study also shows that the combination of Omp purification methods certainly contributes the sensitivity of Omp detection. [source] Insights into the membrane proteome of rat liver peroxisomes: Microsomal glutathione-S-transferase is shared by both subcellular compartmentsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Markus Islinger Dr. Abstract Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl- n -hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation. [source] Evidence for chloroplast control of external Ca2+ -induced cytosolic Ca2+ transients and stomatal closureTHE PLANT JOURNAL, Issue 6 2008Hironari Nomura Summary The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca2+ ([Ca2+]ext)-induced [Ca2+]cyt transients and stomatal closure in Arabidopsis. CAS (Ca2+ sensing receptor) is a plant-specific putative Ca2+ -binding protein that was originally proposed to be a plasma membrane-localized external Ca2+ sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca2+ -binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca2+. In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca2+. Furthermore, using the transgenic aequorin system, we showed that [Ca2+]ext -induced [Ca2+]cyt transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca2+]ext -induced [Ca2+]cyt transients and stomatal closure. [source] | |||||||||||||