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Results for “proteome mapping”

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At least 217 records · Page 12Linked to original sources
PubMed2004

Proteomic analysis of adipocyte differentiation: Evidence that alpha2 macroglobulin is involved in the adipose conversion of 3T3 L1 preadipocytes.

Adipogenesis is an important aspect of energy homeostasis. Here we have used a differential proteome mapping strategy to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3 L1 preadipocytes. Two-dimensional gel electrophoresis analysis identified 8 proteins that are induced following hormone-evoked differentiation. In addition, we found that a alpha2 macroglobulin fragment was abundantly present in 3T3 L1 preadipocytes, but was virtually undetectable in fully differentiated adipocytes. Metabolic radiolabeling with (35S)methionine and Northern blot analysis indicated that the intracellular alpha2 macroglobulin fragment in preadipocytes was derived from the extracellular culture medium, not de novo synthesis. Incubation of preadipocytes with an antialpha2 macroglobulin polyclonal antibody caused depletion of the intracellular alpha2 macroglobulin fragments, and also enhanced spontaneous adipose conversion. These results suggest that intracellular alpha2 macroglobulin fragment inhibits adipocyte differentiation, and that hormone treatment induces differentiation at least in part by suppression of intracellular alpha2 macroglobulin activity in 3T3 L1 preadipocytes.

3T3 Cells↗
PubMed2005

Proteomic analysis of differently expressed proteins in a mouse model for allergic asthma.

Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Many proteins involved in allergic asthma have been identified individually, but complete protein profiles (proteome) have not yet been reported. Here we have used a differential proteome mapping strategy to identify tissue proteins that are differentially expressed in mice with allergic asthma and in normal mice. Mouse lung tissue proteins were separated using two-dimensional gel electrophoresis over a pH range between 4 and 7, digested, and then analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS). The proteins were identified using automated MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. This approach identified 15 proteins that were differentially expressed in the lungs of mice with allergic asthma and normal mice. All 15 proteins were identified by MS, and 9 could be linked to asthma-related symptoms, oxidation, or tissue remodeling. Our data suggest that these proteins may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy.

Animals↗
PubMed2005

Comparative proteomic analysis of peripheral blood eosinophils from healthy donors and atopic dermatitis patients with eosinophilia.

Atopic dermatitis (AD) is an allergic disease that has recently shown a dramatic increase of incidence in developed countries. Eosinophilia, the accumulation of eosinophils, occurs in AD patients through an anti-apoptotic mechanism. To understand the target proteins involved in the anti-apoptotic signaling of eosinophilia, we used a proteomic approach to analyze eosinophil proteins from AD patients with eosinophilia and healthy donors. Protein spots in two-dimensional electrophoresis (2-DE) gels were identified with peptide mass fingerprinting (PMF) based on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and database searching. More spots were observed in the 2-DE proteome map from AD patient samples (1310 +/- 58 spots) than in those from healthy donors (1121 +/- 40 spots). We identified 51 proteins affected by eosinophilia: 19 related to signaling, 8 involved in regulation of metabolism, 4 related to apoptosis, and 3 involved in inflammation. The other identified proteins were associated with transcription, RNA processing, translation, the cytoskeleton, and unknown functions. Among the identified proteins, we observed prominent increases in the expressions of cyclinA2, voltage-dependent anion channel protein 2, and 38 kDa FK506 binding protein 8 in eosinophils from AD patients in comparison to healthy donors. PMF and immunoblotting of a single spot that was expressed in eosinophils from healthy individuals but not in AD patients identified the protein as phosphorylated growth receptor binding 7 (Grb7) adaptor protein. Increased phosphorylation of Grb7 and its upstream signaling protein, focal adhesion kinase (FAK), was detected in low viability eosinophils such as those from healthy donors or in cultured eosinophils (AML14.3D10 cells) treated with dexamethasone. These results suggest that phosphorylation of Grb7 and the expressions of cyclinA2, voltage-dependent anion channel protein 2, and 38 kDa FK506 binding protein 8 may be related with the anti-apoptosis mechanism of eosinophilia.

Apoptosis↗
PubMed2006

Differential proteome analysis of tonsils from children with chronic tonsillitis or with hyperplasia reveals disease-associated protein expression differences.

A proteomic approach has been used to establish a proteome map and differentiate between the protein composition of tonsils from patients with chronic tonsillitis (CT) and that of tonsils with hyperplasia (HPL). Two-dimensional gel analysis was performed with material from four patients with HPL and five patients with CT. An average of approximately 600 spots were detected in each gel. A total of 127 different proteins were identified in 158 spots analyzed by mass spectrometry. Our study revealed disease-associated differences between protein abundance for two protein spots, an HSP27 isoform and UMP-CMP kinase. Both protein spots were more abundant in the CT group. HSP27 ELISA was performed for 32 patients, 12 belonging to the HPL group and 20 to the CT group. ELISA could not be used to differentiate HSP27 isoforms nor to distinguish CT from HPL. HSP27 was found to migrate to two further protein spots in the 2D gels. The differently expressed HSP27 isoform migrated as the most acidic of all the HSP27 isoforms detected, indicating the highest degree of phosphorylation. The sum of all three HSP27 abundances in the gels from the CT group was not different from that of the HPL group, consistent with the ELISA results. Our results suggest that phosphorylation differences caused the observed migration differences of HSP27. Together with the UMP-CMP kinase abundance differences, we conclude that kinase and/or phosphatase activity are different in CT and HPL.

Child↗
PubMed2004

Differential proteome analysis and mass spectrometric characterization of germ line development-related proteins of Caenorhabditis elegans.

Proteome maps and differences of protein patterns of the synchronized larval stage L4 of the temperature-sensitive Caenorhabditis elegans (C. elegans) glp-1 mutant (e2144ts) were investigated after cultivation at 15 degrees C (developing a normal phenotype) or 25 degrees C (developing a mutated phenotype) by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. From the 183 identified protein spots six proteins were found differently expressed. The Vit-6 vitellogenin (CE28594), the hypothetical 17.2 protein (CE25224), the hypothetical 17.4 protein (CE16999), and the heat shock protein 16 kDa (CE14249) were more abundant when growing worm cultures at 25 degrees C. By contrast, the nucleoside diphosphate kinase (CE09650) was found increased at 15 degrees C. Most notably, the eukariotic initiation factor 5A-1 (CE00503), highly abundant at 15 degrees C, was not present in cultures grown at 25 degrees C. Its absence at 25 degrees C can not be attributed to lack of the enzymatic machinery that is necessary for hypusinylation. Instead, a direct downstream effect of the lack of functionality of GLP-1 may cause the expression of this protein. The yolk proteins 115 kDa and 88 kDa were attributed by mass spectrometric protein structure analysis as C-terminal and N-terminal fragments of the Vit-6 vitellogin protein (CE28594), respectively. The cleavage site between both derivatives was located between R764 and A768. A conflict in the database sequences at amino acid positions 1622 and 1623 of vitellogenin-6 was solved by mass spectrometric sequence analysis. The combination of 2-DE with mass spectrometry enabled the identification of mutation-associated differences on somatic gonadal cell and germ line cell development-associated proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis.

Compared to soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on two-dimensional (2-D) gels. One possibility is that many hydrophobic proteins are simply not extracted from the sample prior to 2-D gel separation. We attempted to isolate hydrophobic proteins from Escherichia coli by extracting with organic solvents, then reconstituting the extracted proteins in highly solubilising sample solution amenable to 2-D electrophoresis using immobilized pH gradients (IPGs). This was conducted by an extraction with a mixture of chloroform and methanol, followed by solubilisation using a combination of urea, thiourea, sulfobetaine detergents and tributyl phosphine. Peptide mass fingerprinting assisted in the identification of 13 proteins, 8 of which have not previously been reported on 2-D gels. Five of these new proteins possess a positive hydropathy plot. These results suggest that organic solvent extractions may be useful for selectively isolating some proteins that have previously been missing from proteome maps.

Bacterial Proteins↗
PubMed2001

Two-dimensional electrophoresis and peptide mass fingerprinting of bacterial outer membrane proteins.

Many bacterial outer membrane proteins (OMPs) are missing from two-dimensional (2-D) gel proteome maps. Recently, we developed a technique for 2-D electrophoresis (2-DE) of Escherichia coli OMPs using alkaline pH incubation for isolation of OMPs, followed by improved solubilization conditions for array by 2-DE using immobilized pH gradients. In this report, we expanded our study, examining protein components from the outer membranes of two enteric bacteria, Salmonella typhimurium and Klebsiella pneumoniae (also known as Klebsiella aerogenes), as well as the unrelated, free-living alpha-proteobacteria Caulobacter crescentus. Patterns of OMPs expression appeared remarkably conserved between members of the Enterobacteriaceae, while C. crescentus was unique, displaying a greater number of clusters of higher-molecular-weight proteins (>80 kDa). Peptide mass fingerprinting (PMF) was used for protein identification, and despite matching across-species boundaries, proved useful for first-pass protein assignment of enteric OMPs. In contrast, identification of C. crescentus OMPs was successful only when searching against its recently completed genome. For all three microorganisms examined, the majority of proteins identified on the 2-D gel appear localized to the outer membrane, a result consistent with our previous finding in Escherichia coli. In addition, we discuss some of the benefits and limitations of PMF in cross-species searching.

Bacterial Outer Membrane Proteins↗
PubMed2001

Substituting Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis buffers improves the resolution of focusing patterns.

In a new area of postgenomics challenges, the optimization of protein identification has become a central goal in microbiochemistry. In this work, we demonstrate that the substitution of Coomassie Brilliant Blue for bromophenol blue in two-dimensional electrophoresis (2-DE) buffers improves the focusing of whole proteins from Pseudomonas aeruginosa. This improvement of focusing concerns more particularly basic proteins. This enhancement may be attributed to a better transfer from the first to the second dimension, which probably highlights an increase in the solubility of proteins in the IPG strips. Hence, the use of an efficient tracking dye in the 2-DE buffers may enlarge protein recovery on proteome maps.

Bacterial Proteins↗
PubMed2002

The high sulphur proteins of wool: Towards an understanding of sheep breed diversity.

High sulphur proteins (HSPs) form part of the matrix surrounding the intermediate filaments in the cortical cells of the wool fibre. There are three known families of HSPs, comprising in excess of 40 components and their molecular weights range from 10-30 kDa. Here we report the use of the increased resolving power of isoelectric focusing in the first dimension of two-dimensional electrophoresis and modern gel comparison software to investigate the nature of within- and between-breed variations amongst the proteins of three breeds of sheep: Merino, Romney and Corriedale. In agreement with past studies we observed very little variation in the intermediate filament protein content in wool, both between and within these three sheep breeds. Instead, most of the observed variation occurred among the HSPs, along with some minor variation among the high glycine-tyrosine proteins. Breed-specific differences were observed in the HSP patterns in the wool proteome maps. Merino sheep were found to exhibit the simplest HSP expression patterns, with eight major spots linked to form four pairs. In contrast, the Romney and Corriedale HSP patterns exhibited more spots at lower isoelectric point values (around 4.8), while some of the lower molecular weight HSPs were less prevalent in Romney sheep and absent from the Corriedales.

Animals↗
PubMed2005

Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis.

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.

Chloroform↗
PubMed2005

Proteomic analysis of protein expression profiles during Caenorhabditis elegans development using two-dimensional difference gel electrophoresis.

Coordinated protein expression is critical for the normal execution of animal development. To obtain overall proteome profiles during animal development, a small free-living soil nematode, Caenorhabditis elegans, was used as a model and the developmental changes of protein expressions were analyzed using two-dimensional difference gel electrophoresis. Protein samples from six developmental stages were prelabeled with fluorescent cyanine dyes and separated on two-dimensional electrophoresis gels. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the developmental expression profiles of 231 spots representing 165 proteins. About a quarter of the identified proteins were expressed in multiple spots with different isoelectric points, suggesting a certain proportion of proteins were variously modified. This notion was supported by the observation that about a third of the multispot proteins were stained positive for a phosphoprotein specific dye. While a fairly large number of the proteins showed little alteration in their expression profiles during development, about 40 proteins were found to be significantly either up- or down-regulated between the embryos and newly hatched L1 larvae. Down-regulated proteins included those related to the cell cycle such as MCM-7, PCN-1, and the mitotic checkpoint protein, while up-regulated proteins included structural proteins such as actins, LEV-11, DIM-1, VAB-21, metabolic enzymes such as ATP synthase, ALH-12, fluctose-1,6-bisphosphate aldolase and GPD-3, and galectins. A standard proteome map was obtained where the defects in the mutations of developmental genes and the effects of reagents on the development in C. elegans were analyzed.

Amino Acid Sequence↗
PubMed2006

Low temperature-induced systems failure in Escherichia coli: insights from rescue by cold-adapted chaperones.

The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures.

Adaptation, Physiological↗
PubMed2001

Proteomics of gluten: mapping of subunit 1 Ax2* in Cheyenne cultivar by matrix-assisted laser desorption/ionization.

This paper reports results on the verification of the 1Ax2* high molecular weight glutenin subunit sequence in Cheyenne cultivar. The gene sequence of the protein is known but recently some text changes have been made, and furthermore until now no characterization of post-translational modifications has been reported. The two published sequences, named I and II, differ in four residues at positions 23, 208, 475, and 611. The first sequence contains 20 Arg and 6 Lys residues, producing 26 tryptic fragments, since the Arg(109)-Pro(110) bond is generally not cleaved by trypsin. The second sequence contains 19 Arg and 6 Lys residues, producing 25 tryptic peptides, again because of the Arg(109)-Pro(110) bond. Both sequences generate two cyanogen bromide fragments. Matrix-assisted laser desorption/ionization analysis of the tryptic digest of the high-MW glutenin subunit 1Ax2* resulted in the identification of 24 out of the 26 expected peptides for sequence I, a sequence coverage of 99.5%. These results were sufficient to rule out sequence II and any protein glycosylation and any other post-translational modifications to within the detection limits of the method. It was found that the choice of matrix considerably influenced the sequence coverage in peptide mapping.

Amino Acid Sequence↗
PubMed2004

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins.

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.

Animals↗
PubMed2004

Efficacy and compatibility with mass spectrometry of methods for elution of proteins from sodium dodecyl sulfate-polyacrylamide gels and polyvinyldifluoride membranes.

The resolving power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with isoelectric focusing in two-dimensional gel electrophoresis has made it one of the most important techniques for resolving complex mixtures, and it is of great importance for proteome mapping projects. As a result of this, methods for postelectrophoretic protein characterization are of great interest as exemplified by in situ protease digestion combined with mass spectrometry (MS), which is the method of choice for identification of proteins. In this study we have developed and compared methods for recovering intact proteins from polyacrylamide gels and electroblotting membranes to define efficient methods compatible with MS. These methods complement in situ digestion protocols and allow determination of the molecular mass of whole proteins separated by SDS-PAGE. Passive elution of proteins from SDS-PAGE gels was efficient only in the presence of SDS, whereas electroelution was achieved using buffers without SDS. Surface-enhanced laser desorption/ionization MS (SELDI-MS) analysis of proteins eluted in the presence of SDS was possible using ion exchange ProteinChip arrays for concentration of sample and removal of SDS. Comparison of different electroblotting methods verified that the different membranes and buffers were equally efficient for transfer of proteins in the range 20-100 kDa. Elution from polyvinyldifluoride membranes was most efficient using either concentrated solutions of trifluoroacetic acid (TFA) or combinations of 8M urea and 1% Triton X-100, 1% Tween 20, or 40% isopropanol. The same result was obtained using nitrocellulose membranes, except that these were incompatible with organic solvent and TFA. Elution by TFA was compatible with matrix-assisted laser desorption/ionization MS (MALDI-MS) but was complicated by a high degree of trifluoroacetylation of the proteins. Alternatively, elution by 8M urea+1% Triton X-100, 1% Tween 20, or 40% isopropanol was compatible with both SELDI-MS and MALDI-MS. Eluted proteins were identified in MS experiments by intact mass determination, by peptide mapping, and by MS/MS analysis.

Acrylic Resins↗
PubMed2004

The genomics of malaria infection.

Malaria research is now dominated by information flowing from the genome sequencing projects and the associated transcriptome- and proteome-mapping projects. As more species are sequenced, comparative and phylogenetic comparisons are improving the quality of gene finding, and are providing various approaches to the identification of genes important to parasite biology and the pathogenesis of disease. We are still in the early days of exploiting these data in a systematic way and the sheer volume of data presents daunting challenges. This article reviews the progress in using this genomic information and discusses opportunities for other approaches.

Animals↗
PubMed2004

Anti-stress activity of Propionibacterium freudenreichii : identification of a reactivative protein.

Propionibacterium freudenreichii subsp. shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells. It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect. In order to characterise this effect at the molecular level, we have purified the active component from a P. freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography. The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase. The latter was localised in the P. freudenreichii proteomic map. It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses. The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.

4-Nitroquinoline-1-oxide↗