Isolation protocol for two-dimensional-polyacrylamide gel electrophoresis analysis of Haloferax volcanii proteome.
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Algorithms↗
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Results for “Comparative proteomics”
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Changes in magnetization transfer MRI correlate with spreading depression-induced astroglial reactivity and increased protein expression in mice.
OBJECTIVE: Gliosis refers to a range of glial cell transformations that vary according to specific brain pathologic states. Disease, however, is not a prerequisite for gliosis because glial reactivity may also be seen in regions of increased physiologic activity. Our study tests the hypothesis that high-field-strength magnetization transfer MRI is a sensitive technique for detecting transient glial reactivity after experimental spreading depression, a relatively benign perturbation unaccompanied by cell injury. MATERIALS AND METHODS: Unilateral neocortical spreading depression was elicited in mouse cerebral hemispheres and confirmed by transcranial blood flow and extracellular potential measurements. After 3 days, mice were imaged at 4 T using magnetization transfer techniques. Astroglial reactivity was determined immunohistochemically, and protein expression in control and experimental hemispheres was compared using proteomic techniques. RESULTS: Sixteen ([mean +/- SD] +/- 3) spreading depressions (n = 10) were recorded in experimental hemispheres. Spreading depression was never observed in control hemispheres. At 3 days, an 8% decrease (p < 0.05, n = 4) in magnetization transfer signal intensity was measured in experimental hemispheres, which was associated with a 37% increase (p < 0.001, n = 4) in the intensity of glial fibrillary acidic protein staining. Proteomic analysis performed 3 days after the induction of spreading depression showed upregulation of at least 56 proteins, including extracellular and intracellular elements. CONCLUSION: Magnetization transfer at 4.0-T MRI is a sensitive method for detecting glial reactivity and changes in protein expression not associated with cell injury. These results suggest magnetization transfer MRI techniques may have potential for detecting glial reactivity in physiologic processes such as learning and in early disease states.
Animals↗
[Proteomic analysis of different fractions in M. tuberculosis culture].
To provide a basis of molecular genetic analysis of M. tubereulosis, the proteomic profiling was prompted. M. tuberculosis H37RV was cultured in Sauton medium at 37 degrees C for 3 weeks, harvested and fractionated into three portions: suspension filtration proteins(A), cytosol proteins(B) and membrane proteins(C). These fractions were analyzed by pH3-10 IPG gradient and SDS-PAGE. The silver-stained technigue and gel images were used. Then the image was transfered into 2-DE gel analysis Software. A part of protein sports of expression level higher from the culture filtrate fraction were identified by peptide mass fingerprinting. A total of 907, 884 and 681 protein sports were observed for A. B. C fractions in M. tuberculosis H37RV, respectively. Distribution of proteins mass for 3 fractions were principally similar, About 70.5-74.4 per cent were distributed in the ranges of Mr 10-49 kD.pI of the proteins for A, B fractions were pricipally similar, About 80.9-83.5 per cent were distributed in the ranges of pH 3.0-6.4, But the number of protein sports for C fraction distributed in the ranges of pH 7.6-10.0 were more than A, B fractions. The number of protein sports of expression level higher for A, B and C fractions were 71(7.8%), 242(27.4%), 19 (2.8%), respectively. 90 pen cent from them, pH of the proteins were distributed in the ranges of pH 3.0-6.4. 73.1 per cent for proteins mass of C fraction were distributed in the ranges of 10 kD-49 kD, which were more than A, B fractions. Nine of the proteins identified ih this study appeared to be homology or putative fanction proteins, but another five proteins were unknown. The proteomic profiling of different fractions in M. tuberculosis obtained in here will be provide a basis for detailed analysis of biology functions of the proteins.
Bacterial Proteins↗
Proteomics reveals effects effects of aquaculture on fish.
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Animals↗
[Analysis of the different proteomes between the acute leukemia cells and normal white blood cells].
This study was aimed to analyze the different proteomes between human acute leukemia (AL) cells and normal white blood cells by proteomic technology in order to lay the basis for diagnosing AL and understanding the mechanism of leukemogenesis. The proteins from AL cells of 40 AL patients identified by FAB classification and proteins from normal lymphocytes and granulocytes of 20 normal volunteers were separated by two-dimensional electrophoresis (2-DE), and the differentially expressed proteins between the two groups were identified by both matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electronspray ionization (ESI)-MS/MS. The results showed that among the differentially expressed proteins between AL cells and normal lymphocytes and granulocytes, some proteins involved in the process of malignant transformation (such as Op18, NM23-H1), cell proliferation (such as PCNA) and apoptosis inhibition (such as tumor necrosis factor inhibitor protein) were found to be up regulated in AL cells. However, some proteins involved in differentiation and physiological functions of normal cells were down regulated in AL cells. It is concluded that there are many events involved in the process of leukemogenesis, expression of some proteins relating to the malignant transformation, cell proliferation and apoptosis inhibition are up-regulated in AL cells. The proteome analysis may provide a new approach to explaining the molecular mechanism underlying the pathogenesis of AL.
Adolescent↗
Proteomic identification of potential susceptibility factors in drug-induced liver disease.
Drug-induced liver disease (DILD) causes significant morbidity and mortality and impairs new drug development. Currently, no known criteria can predict whether a drug will cause DILD or what risk factors make an individual susceptible. Although it has been shown in mouse studies that the disruption of key regulatory factors, such as cyclooxygenase-2 (COX-2), interleukin (IL)-6, and IL-10, increased susceptibility to DILD caused by acetaminophen (APAP), no single factor seems to be absolute. As an approach to better understand the multifactorial basis of DILD, we compared the hepatic proteome of mice that are resistant (SJL) and susceptible (C57Bl/6) to APAP-induced liver disease (AILD), using solution-based isotope-coded affinity tag (ICAT) liquid chromatography mass spectrometry. Several novel factors were identified that were more highly expressed in the livers of SJL mice, including those involved in stress response, cell proliferation and tissue regeneration, and protein modification, implicating these proteins as potential hepatoprotective factors. There was also a selective loss of several mitochondrial proteins from the livers of the susceptible C57Bl/6 mice, suggesting that the loss of functional mitochondria may indeed play a role in AILD. These findings indicate that comparative hepatic proteomic analyses of susceptible and resistant mouse strains may provide a global approach for identifying potential risk factors and mechanistic pathways responsible for DILD.
Acetaminophen↗
Identification of ethylene-mediated protein changes during nodulation in Medicago truncatula using proteome analysis.
Ethylene has been hypothesised to be a regulator of root nodule development in legumes, but its molecular mechanisms of action remain unclear. The skl mutant is an ethylene-insensitive legume mutant showing a hypernodulation phenotype when inoculated with its symbiont Sinorhizobium meliloti. We used the skl mutant to study the ethylene-mediated protein changes during nodule development in Medicago truncatula. We compared the root proteome of the skl mutant to its wild-type in response to the ethylene precursor aminocyclopropane carboxylic acid (ACC) to study ethylene-mediated protein expression in root tissues. We then compared the proteome of skl roots to its wild-type after Sinorhizobium inoculation to identify differentially displayed proteins during nodule development at 1 and 3 days post inoculation (dpi). Six proteins (pprg-2, Kunitz proteinase inhibitor, and ACC oxidase isoforms) were down-regulated in skl roots, while three protein spots were up-regulated (trypsin inhibitor, albumin 2, and CPRD49). ACC induced stress-related proteins in wild-type roots, such as pprg-2, ACC oxidase, proteinase inhibitor, ascorbate peroxidase, and heat-shock proteins. However, the expression of stress-related proteins such as pprg-2, Kunitz proteinase inhibitor, and ACC oxidase, was down-regulated in inoculated skl roots. We hypothesize that during early nodule development, the plant induces ethylene-mediated stress responses to limit nodule numbers. When a mutant defective in ethylene signaling, such as skl, is inoculated with rhizobia, the plant stress response is reduced, resulting in increased nodule numbers.
Amino Acid Sequence↗
Proteomics of Arabidopsis seed germination. A comparative study of wild-type and gibberellin-deficient seeds.
We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component alpha-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for the ga1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a beta-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.
Arabidopsis↗
[Identification of AGR2 protein, a novel potential cancer marker, using proteomics technologies].
A comparative analysis of the proteins in prostate tissues of the patients operated for hyperplasia (n = 7) or cancer (n = 5) was performed aiming to search for protein diagnostic markers. Differences in several minor proteins were detected using two-dimensional electrophoresis according to O'Farrel; among them, an additional protein with a molecular weight of 19 kDa and an isoelectric point of 9.0 was observed in four of the cancer cases. Mass spectrometry allowed this protein to be identified as the androgen-induced secreted protein AGR2. The possibility of using AGR2 as a diagnostic marker of prostate cancer is discussed.
Biomarkers, Tumor↗
On 3-D graphical representation of proteomics maps and their numerical characterization.
We consider numerical characterization of proteomics maps by representing a map as a three-dimensional graphical object based on x, y coordinates of the spots and using their relative abundance as the z coordinate. In our representation the protein spots are first ordered based on their relative abundance and labeled accordingly. In the next step a 3-D path is constructed connecting spots having adjacent labels. Finally a matrix is constructed by assigning to each pairs of labels (i, j) matrix element, the numerical value of which is based on the quotients of the Euclidean distance and the distance along the 3-D zigzag between the two points. The approach has been illustrated on a fragment of a proteomics map and compared with 2-D graphical representation of proteomics maps.
Computer Graphics↗
Shotgun proteomics using the iTRAQ isobaric tags.
Shotgun proteomic methods involving isobaric tagging of peptides enable high-throughput proteomic analysis. iTRAQ reagents allow simultaneous identification and quantitation of proteins in four different samples using tandem mass spectrometry (MS). In this article, we provide a brief description of proteome analysis using iTRAQ reagents and review the current applications of these reagents in proteomic studies. We also compare different aspects of protein identification including protein sequence coverage and proteome coverage obtained using iTRAQ reagents with those using other shotgun proteomic techniques. We briefly discuss the issue of isotope purity correction in measured peak areas during protein quantitation using iTRAQ reagents. Finally, we conclude with some of the current challenges in MS-based proteomic analysis that are limiting protein identifications obtained by different shotgun proteomic methods.
Animals↗
Identification of fibrosis-relevant proteins using DIGE (difference in gel electrophoresis) in different models of hepatic fibrosis.
Proteomics became a more and more important technique for the large-scale analysis of proteins during the last years. Two-dimensional (2D) electrophoresis as a major tool of proteomics is a powerful method to compare two different biological stages (e. g. healthy and diseased tissue) and to find differences in their protein pattern. One major problem in proteomics is the gel to gel variation of two-dimensional gel electrophoresis, which could cause artefacts in the detection of expression differences. The "difference in gel electrophoresis" (DIGE) technique allows the separation of two proteomes in the same gel. The protein pools were labelled with different fluorescent dyes and equal amounts of protein were separated in the same gel. Another advantage of DIGE is the possibility to separate an internal standard labelled with a third dye in the same gel to allow quantitative expression analysis. We compared proteomes of three different fibrosis models with the appropriate control (tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing HepG2 cells in comparison to a HepG2 control, freshly isolated HSC in comparison to activated HSC and healthy mouse liver in comparison to fibrotic mouse liver). Among the differentially expressed proteins several were already found to be relevant for fibrosis but we also detected some proteins like the selenium binding protein 2 which might be relevant for hepatic fibrosis.
Animals↗
Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.
BACKGROUND: The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. RESULTS: We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. CONCLUSIONS: By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.
Amino Acid Sequence↗
Proteomic analysis of hemangioblastoma cyst fluid.
OBJECTIVE: Hemangioblastomas are benign CNS tumors that occur sporadically or in patients with von Hippel-Lindau (VHL) disease. These tumors are characteristically associated with formation of intra- or peritumoral cysts. Hemangioblastoma cyst formation is a major cause of morbidity and mortality with these tumors. While peritumoral cysts have been suggested to result from vascular leakage, the mechanism of intratumoral cyst formation is not understood. METHODS: To elucidate the origin of intratumoral hemangioblastoma cyst fluid, we characterized its biochemical composition by two-dimensional (2D) proteomic profiling followed by sequencing of several proteins. The proteomic pattern of intratumoral cyst fluid was furthermore compared to the proteomic pattern of serum, hemangioblastoma tumor tissue, and hemangioblastoma peritumoral cyst fluid. RESULTS: We show that proteomic patterns of intra- and peritumoral cyst fluid are identical Both are highly similar to serum and not to tumor. CONCLUSIONS: Intratumoral hemangioblastoma cyst fluid originates from serum. Cyst formation associated with hemangioblastoma, whether peri- or intratumoral, is a consequence of vascular leakage. Anti-VEGF therapy may effectively control hemangioblastoma cyst formation.
Biomarkers, Tumor↗
Comparative analysis of the heat stable proteome of radicles of Medicago truncatula seeds during germination identifies late embryogenesis abundant proteins associated with desiccation tolerance.
A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting alpha helices and beta sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.
Desiccation↗
Comparative analysis of phosphoprotein-enriched myocyte proteomes reveals widespread alterations during differentiation.
The differentiation of skeletal muscle has been associated with altered phosphorylation status of individual proteins. However, a global analysis of protein phosphorylation during myogenesis has yet to be undertaken. Here, we report the identification of over 130 putative phosphoproteins from murine C2C12 muscle cells. Cell extracts were fractionated on phosphoprotein enrichment columns and the resulting proteins were detected by two-dimensional gel electrophoresis and silver stain, and identified by liquid chromatography coupled to electrospray tandem mass spectrometry. The early differentiation of C2C12 myoblasts was found to be accompanied by changes in the phosphorylation or expression of numerous proteins including cytoskeletal, heat shock and signaling proteins, the pp32 family of nuclear phosphoproteins, several disease-associated gene products and other characterized and uncharacterized proteins.
Cell Differentiation↗
Global analyses of transcriptomes and proteomes of a parent strain and an L-threonine-overproducing mutant strain.
We compared the transcriptome, proteome, and nucleotide sequences between the parent strain Escherichia coli W3110 and the L-threonine-overproducing mutant E. coli TF5015. DNA macroarrays were used to measure mRNA levels for all of the genes of E. coli, and two-dimensional gel electrophoresis was used to compare protein levels. It was observed that only 54 of 4,290 genes (1.3%) exhibited differential expression profiles. Typically, genes such as aceA, aceB, icdA, gltA, glnA, leu operon, proA, thrA, thrC, and yigJ, which are involved in the glyoxylate shunt, the tricarboxylic acid cycle, and amino acid biosynthesis (L-glutamine, L-leucine, proline, and L-threonine), were significantly upregulated, whereas the genes dadAX, hdeA, hdeB, ompF, oppA, oppB, oppF, yfiD, and many ribosomal protein genes were downregulated in TF5015 compared to W3110. The differential expression such as upregulation of thr operon and expression of yigJ would result in an accumulation of L-threonine in TF5015. Furthermore, two significant mutations, thrA345 and ilvA97, which are essential for overproduction of L-threonine, were identified in TF5015 by the sequence analysis. In particular, expression of the mutated thrABC (pATF92) in W3110 resulted in a significant incremental effect on L-threonine production. Upregulation of aceBA and downregulation of b1795, hdeAB, oppA, and yfiD seem to be linked to a low accumulation of acetate in TF5015. Such comprehensive analyses provide information regarding the regulatory mechanism of L-threonine production and the physiological consequences in the mutant stain.
Electrophoresis, Gel, Two-Dimensional↗
Drug induced proteome changes in Candida albicans: comparison of the effect of beta(1,3) glucan synthase inhibitors and two triazoles, fluconazole and itraconazole.
The dimorphic fungus Candida albicans is an opportunistic human pathogen. Candidiasis is usually treated with azole antifungal agents. However clinical treatments may fail due to the appearance of resistance to this class of antifungal agents in Candida. Echinocandin derivatives are an alternative for the treatment of these fungal infections and are active against azole resistant isolates of C. albicans. Azoles inhibit the lanosterol 14 alpha demethylase which is a key enzyme in the synthesis of ergosterol. In contrast, the echinocandin class of antibiotics inhibit noncompetitively beta-(1,3)-D-glucan synthesis in vitro. We have investigated the impact of mulundocandin on the proteome of C. albicans and compared it to those of a mulundocandin derivative, as well as to two azoles of different structure, fluconazole and itraconazole. The changes in gene expression underlying the antifungal responses were analyzed by comparative 2-D PAGE. Dose dependant responses were kinetically studied on C. albicans grown at 25 degrees C (yeast form) in synthetic dextrose medium. This study shows that antifungals with a common mechanism of action lead to comparable effects at the proteome level and that a proteomics approach can be used to distinguish different antifungals, with the promise to become a useful tool to study drugs of unknown mechanism of action.
Antifungal Agents↗