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1.
The urchin Paracentrotus lividus has been characterized via previous capture and enhancement of low-abundance proteins with combinatorial peptide ligand libraries (CPLL, ProteoMiner). Whereas in the control only 26 unique gene products could be identified, 82 species could be detected after CPLL treatment. Due to the overwhelming presence of two major proteins-the toposome (a highly glycosylated, modified calcium-binding, iron-less transferrin) and the major yolk proteins, belonging to the class of cell adhesion proteins-which constituted about 70% of the proteome of this biological fluid and strongly interfered with the capture of the minority proteome, no additional proteins could be detected. Yet, at present, this constitutes the most thorough investigation of the proteome of this biological fluid. 相似文献
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3.
Rod J.Snowdon 《Acta Genetica Sinica》2010,(5)
The diploid species Brassica rapa(genome AA)and B.oleracea(genome CC)were compared by full-scale proteome analyses of seedling.A total of 28.2% of the proteins was common to both species,indicating the existence of a basal or ubiquitous proteome.However,a number of discriminating proteins(32.0%)and specific proteins(39.8%)of the Brassica A and C genomes,respectively,were identified,which could represent potentially species-specific functions.Based on these A or C genome-specific proteins,a number of PCR-bas... 相似文献
4.
Peptide patterns and immunological properties of the cytoplasmic and chloroplastic -1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves have been studied and were compared with those of phosphorylases from other sources. The two spinach leaf phosphorylases were immunologically different; a limited cross-reactivity was observed only at high antigen or antibody concentrations. Peptide mapping of the two enzymes resulted in complex patterns composed of more than 20 fragments; but no peptide was electrophoretically identical in both proteins. Approximately 13 to 15 of the fragments exhibited antigeneity but no cross-reactivity of any peptide was observed. Therefore, the two compartment-specific phosphorylase forms from spinach leaves represent isoenzymes possessing different primary structures. Peptide patterns of potato tuber and rabbit muscle phosphorylase were different from those of the two spinach leaf enzymes. Although the potato tuber phosphorylase resides in the plastidic compartment and is kinetically closely related to the chloroplastic spinach enzyme, it reacted more strongly with the anti-cytoplasmic-phosphorylase immunoglobulin G. Similar results were obtained with rabbit muscle phosphorylase. These observations support the assumption that the chloroplast-specific phosphorylase isoenzyme has a higher structural diversity than does the cytoplasmic counterpart.Abbreviations EDTA
ethylenediaminetetraacetic acid
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- PEG
polyethylene glycol (approx. MW 8000)
I=Schächtele and Steup 1986 相似文献
5.
The diploid species Brassica rapa(genome AA)and B.oleracea(genome CC)were compared by fuU-seale proteome analyses of seedling.A total of 28.2% of the proteins was common to both species,indicating the existence of a basal or ubiquitous proteome.How-ever,a number of discriminating proteins(32.0%)and specific proteins(39.8%)of the Brassica A and C genomes,respectively,were identified,which could represent potentially species-specific functions.Based on these A or C genome-specific proteins,a number of PCR-based markers to distinguish B.rapa and B.oleracea species were also developed. 相似文献
6.
Sivakumar Prasanth Kumar Vilas R. Parmar Yogesh T. Jasrai Himanshu A. Pandya 《Journal of chemical biology》2015,8(3):95-105
Kinetin, a cytokinin which promotes seed germination by inhibiting the action of abscisic acid, is an important molecule known to trigger various molecular mechanisms by interacting with an array of proteins shown from experimental observations in various model organisms. We report here the prediction of most probable protein targets of kinetin from spinach proteome using in silico approaches. Inverse docking and ligand-based similarity search was performed using kinetin as molecule. The former method prioritized six spinach proteins, whereas the latter method provided a list of protein targets retrieved from several model organisms. The most probable protein targets were selected by comparing the rank list of docking and ligand similarity methods. Both of these methods prioritized chitinase as the most probable protein target (ΔGpred = 5.064 kcal/mol) supported by the experimental structure of yeast chitinase 1 complex with kinetin (PDB: 2UY5) and Gliocladium roseum chitinase complex with 3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione (caffeine; 3G6M) which bears a 3D similarity of 0.43 with kinetin. An in vitro study to evaluate the effect of kinetin on spinach seed germination indicated that a very low concentration of kinetin (0.5 mg/l) did not show a significant effect compared to control in inducing seed germination process. Further, higher levels of kinetin (>0.5 mg/l) constituted an antagonist effect on spinach seed germination. It is anticipated that kinetin may have a molecular interaction with prioritized protein targets synthesized during the seed germination process and reduces growth. Thus, it appears that kinetin may not be a suitable hormone for enhancing spinach seed germination in vitro.
Electronic supplementary material
The online version of this article (doi:10.1007/s12154-015-0135-3) contains supplementary material, which is available to authorized users. 相似文献7.
Guerrier L Claverol S Fortis F Rinalducci S Timperio AM Antonioli P Jandrot-Perrus M Boschetti E Righetti PG 《Journal of proteome research》2007,6(11):4290-4303
A combinatorial ligand library, composed of millions of diverse hexapeptide baits, able to capture and concentrate the "low-abundance" proteome while drastically cutting the concentration of the most abundant species, has been applied to the exploration of the soluble platelet proteome. Mass spectrometry analysis of untreated and library-treated platelets has resulted in the identification of 435 unique gene products. Of those, 147 entries (35% of the total) have not been described among the list of >1100 proteins in proteomic platelet investigations reported before. In addition, the analysis of excised spots from two-dimensional electrophoresis analysis allowed 57 other proteins to be added that were not found in LC-MS analysis, 33 of them not described before in proteomics studies, bringing the total number of new gene products to 180. Thus, the present data add a non-negligible number of species for continuing the "cartography" of the proteomic asset of platelets, in view of completing the mapping procedure for a deeper understanding of the physiology and pathology of this blood cell. Because the capturing process is performed under physiological conditions, by exploiting, for binding to the combinatorial library, the native protein configuration, the described technique is not adapted to capture highly hydrophobic proteins, which need strong denaturing and solubilizing conditions that are incompatible with our working procedure. Thus, our list reports essentially hydrophilic proteins, with negative GRAVY indexes. 相似文献
8.
Accumulation of oxidatively modified proteins is a hallmark of organismal aging in vivo and of cellular replicative senescence in vitro. Failure of protein maintenance is a major contributor to the age‐associated accumulation of damaged proteins that is believed to participate to the age‐related decline in cellular function. In this context, quantitative proteomics approaches, including 2‐D gel electrophoresis (2‐DE)‐based methods, represent powerful tools for monitoring the extent of protein oxidative modifications at the proteome level and for identifying the targeted proteins, also referred as to the “oxi‐proteome.” Previous studies have identified proteins targeted by oxidative modifications during replicative senescence of human WI‐38 fibroblasts and myoblasts and have been shown to represent a restricted set within the total cellular proteome that fall in key functional categories, such as energy metabolism, protein quality control, and cellular morphology. To provide mechanistic support into the role of oxidized proteins in the development of the senescent phenotype, untargeted metabolomic profiling is also performed for young and senescent myoblasts and fibroblasts. Metabolomic profiling is indicative of energy metabolism impairment in both senescent myoblasts and fibroblasts, suggesting a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts and fibroblasts. 相似文献
9.
The present review deals with the use of combinatorial ligand libraries, composed by hexapeptides, in the capture and concentration of the low‐abundance proteome. This method, first reported in 2005, is compared with other current methodologies aimed at exploring the “deep proteome”, such as: depletion of high‐abundance proteins (especially in sera and cerebrospinal fluid) by individual or combined antibodies (up to 20 against the most abundant species); narrow (1‐pH‐unit) IPGs (zoom gels) and prefractionation with multicompartment electrolyzers or with Off‐Gel electrophoresis. The physico‐chemical properties of the hexapeptide library are also explored, namely in assessing the proper length of the baits and the behavior of shorter oligopeptides, down to capture elicited by single amino acids. A number of examples on the use of this library is given, such as the analysis of biological fluids (human sera, urine, bile, cerebrospinal fluid) and of cell lysates (platelets, red blood cells). In all cases, it was possible to detected from three to five times as many proteins as compared to control, untreated samples. Perhaps the most spectacular results were obtained with the erythrocyte proteome, where 1570 proteins could be identified in the “minority” proteome, representing only 2% of the total cell lysate. Another interesting area of application regards the concentration and detection of trace impurities contaminanting r‐DNA proteins meant for human consumption: several host proteins, never reported before, could be revealed for the first time. Other nonhuman samples are currently under investigation, such as egg‐white (where no less than 148 unique gene products could be identified), egg yolk (with 255 unique species) and latex from Hevea brasiliensis. It is anticipated that the ligand library could be a most useful tool for detecting biomarkers for different pathologies and for drug treatment. As a future outlook, one could envision the synthesis of specialized libraries able to capture given classes of proteins (e.g., phospho‐ and glyco‐proteins). Additionally, the library could be used in association with other techniques currently in vogue, such as zoom gels, Off‐Gels, and the like. 相似文献
10.
Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties. 相似文献
11.
Jiang XS Dai J Sheng QH Zhang L Xia QC Wu JR Zeng R 《Molecular & cellular proteomics : MCP》2005,4(1):12-34
Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location. 相似文献
12.
After 10 years of extensive proteomic research, it has become increasingly apparent that new technologies are sorely needed for detecting the low-abundance proteome-those proteins (up to 50% in any proteome) whose concentration in tissues or cells falls below the detection limits of currently available methodologies. Here we survey one such method: a combinatorial ligand library (called ProteoMiner), comprising dozens of millions of hexapeptides capable of interacting with most, if not all, proteins in any given proteome. They act by drastically reducing the signal of high-abundance species while increasing the level of the low-abundance components to bring their signal within the detection limit of present-day tools. Such a library has been tested against a number of human biological fluids, such as sera, urine, cerebrospinal fluid as well as against cell lysates (e.g., platelets, red blood cells) with interesting results. 相似文献
13.
Park EC Yoon JB Seong JS Choi KS Kong ES Kim YJ Park YM Park EM 《Preparative biochemistry & biotechnology》2006,36(1):19-35
Reactive oxygen species (ROS), generated by ionizing radiation, has been implicated in its effect on living tissues. We confirmed the changes in the oxidative stress markers upon irradiation. We characterized the changes in the proteome profile in rat liver after administering irradiation, and the affected proteins were identified by MALDI-TOF-MS and ESI-MS/MS. The identified proteins represent diverse sets of proteins participating in the cellular metabolism. Our results demonstrated that proteomics analysis is a useful method for characterization of a global proteome change caused by ionizing radiation to unravel the molecular mechanisms involved in the cellular responses to ionizing radiation. 相似文献
14.
The proteome of coconut milk has been extensively mapped via capture at three pH values with combinatorial peptide ligand libraries (CPLL). A grand total of 307 unique gene products could be listed, 200 discovered via CPLL capture, 137 detected in the control, untreated material and 30 species in common between the two sets of data. This is by far the most extensive mapping of coconut milk, in which, up to the present, only a dozen proteins were known, those belonging to the high- to very-high abundance class. The database of coconut contains only 106 proteins: of those, only six are listed in our table. The vast majority of the classified proteins, thus, has been identified only by homologies with sequences deposited in the general viridiplantae database. This unique set of data could be the starting point for nutritionists and researchers involved in nutraceutics for enucleating some proteins responsible for some of the unique beneficial health effects attributed to coconut milk. 相似文献
15.
Roux-Dalvai F Gonzalez de Peredo A Simó C Guerrier L Bouyssié D Zanella A Citterio A Burlet-Schiltz O Boschetti E Righetti PG Monsarrat B 《Molecular & cellular proteomics : MCP》2008,7(11):2254-2269
The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored. Here we used a combinatorial library of hexameric peptides as a capturing agent to lower the signal of hemoglobin and amplify the signal of low to very low abundance proteins in the cytoplasm of human red blood cells (RBCs). Two types of hexapeptide library beads have been adopted: amino-terminal hexapeptide beads and beads in which the peptides have been further derivatized by carboxylation. The amplification of the signal of low abundance and suppression of the signal of high abundance species were fully demonstrated by two-dimensional gel maps and nano-LC-MSMS analysis. The effect of this new methodology on quantitative information also was explored. Moreover using this approach on an LTQ-Orbitrap mass spectrometer, we could identify with high confidence as many as 1578 proteins in the cytoplasmic fraction of a highly purified preparation of RBCs, allowing a deep exploration of the classical RBC pathways as well as the identification of unexpected minor proteins. In addition, we were able to detect the presence of eight different hemoglobin chains including embryonic and newly discovered globin chains. Thus, this extensive study provides a huge data set of proteins that are present in the RBC cytoplasm that may help to better understand the biology of this simplified cell and may open the way to further studies on blood pathologies using targeted approaches. 相似文献
16.
The nucleus is a highly organized organelle with an intricate substructure of chromatin, RNAs, and proteins. This environment represents a challenge for maintaining protein quality control, since non-native proteins may interact inappropriately with other macromolecules and thus interfere with their function. Maintaining a healthy nuclear proteome becomes imperative during times of stress, such as upon DNA damage, heat shock, or starvation, when the proteome must be remodeled to effect cell survival. This is accomplished with the help of nuclear-specific chaperones, degradation pathways, and specialized structures known as protein quality control (PQC) sites that sequester proteins to help rapidly remodel the nuclear proteome. In this review, we focus on the current knowledge of PQC sites in Saccharomyces cerevisiae, particularly on a specialized nuclear PQC site called the intranuclear quality control site, a poorly understood nuclear inclusion that coordinates dynamic proteome triage decisions in yeast. 相似文献
17.
María Jesús Lerma-García Alfonsina D’Amato Elisa Fasoli Ernesto Francisco Simó-Alfonso Pier Giorgio Righetti 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(9):1493-1499
Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteome of a popular aperitif in Northern Italy, called “Amaro Branzi”, stated to be an infusion of a secret herbal mixture, of which some ingredients are declared on the label, namely Angelica officinalis, Gentiana lutea and orange peel, sweetened by a final addition of honey. In order to assess the genuineness of this commercial liqueur, we have prepared extracts of the three vegetable ingredients, assessed their proteomes, and compared them to the one found in the aperitif. The amaro's proteome was identified via prior capture with CPLLs at two different pH values (2.2 and 4.8). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could confirm the presence of the following: six proteins originating from honey, 11 from orange peels, 29 from G. lutea and 46 from A. officinalis (including shared species), plus 33 species which could not be attributed to the other secret ingredients, due to paucity of genomic data on plant proteins, for a total of 93 unique gene products (merging shared proteins). This fully confirmed the genuineness of the product. Considering that most of these species could be present in trace amounts, undetectable by conventional techniques, the CPLL methodology, due to its ability to enhance the signal of trace components up to 3 to 4 orders of magnitude, could represent a powerful tool for investigating the genuineness and natural origin of commercial beverages in order to protect consumers from adulterated products. 相似文献
18.
Ferrari G Garaguso I Adu-Bobie J Doro F Taddei AR Biolchi A Brunelli B Giuliani MM Pizza M Norais N Grandi G 《Proteomics》2006,6(6):1856-1866
We compared the proteome of detergent-derived group B Neisseria meningitidis (MenB) outer membrane vesicles (DOMVs) with the proteome of outer membrane vesicles (m-OMVs) spontaneously released into culture supernatant by MenB delta gna33, a mutant in which the gene coding for a lytic transglycosylase homologous to the E. coli MltA was deleted. In total, 138 proteins were identified in DOMVs by 1- and 2-DE coupled with MS; 64% of these proteins belonged to the inner membrane and cytoplasmic compartments. By contrast, most of the 60 proteins of m-OMVs were classified by PSORT as outer membrane proteins. When tested for their capacity to elicit bactericidal antibodies, m-OMVs elicited a broad protective activity against a large panel of MenB strains. Therefore, the identification of mutations capable of conferring an OMV-releasing phenotype in bacteria may represent an attractive approach to study bacterial membrane composition and organization, and to design new efficacious vaccine formulations. 相似文献
19.
Sabarth N Lamer S Zimny-Arndt U Jungblut PR Meyer TF Bumann D 《The Journal of biological chemistry》2002,277(31):27896-27902
Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane. 相似文献
20.
Castagna A Cecconi D Sennels L Rappsilber J Guerrier L Fortis F Boschetti E Lomas L Righetti PG 《Journal of proteome research》2005,4(6):1917-1930
The human urinary proteome has been reassessed and re-evaluated via a novel concentration/equalization technique, exploiting beads coated with hexameric peptide ligand libraries. These beads act by capturing the whole protein spectra contained in the sample, by drastically reducing the level of the most abundant species, while strongly concentrating the more dilute and rare ones. In a control urine sample, 134 unique proteins could be identified. The first bead eluate (in thiourea, urea, and CHAPS) permitted the identification of 317 gene products, whereas the second eluate (in 9 M urea, pH 3.8) allowed the identification of another 95 unique proteins. By eliminating redundancies, a total of 383 unique gene products could be identified in human urines. This represents a major increment as compared to data reported in recent literature. By comparing our data with those reported to the present, an additional 251 proteins could be added to the list, thus bringing the total unique gene products so far identified in human urines to ca. 800 species. 相似文献