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This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin 1 recepto...
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This paper describes the use of fluorescence two-dimensional differential in-gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin 1 receptor knockout (NK_1R-/-) and wild type (NK_1R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK_1R-/- and NK_1R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two-dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In-gel Analysis software. A Cy2-labeled sample pool was co-electrophoresed with all Cy3- and Cy5-labeled sample pairs as an internal standard providing a link for inter-gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK_1R-/- mice compared to NK_1R+/+ controls. Matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.
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Cancer results from the accumulation of genomic alterations. As the genome is functionally translated to the proteome and regulates tumor cell behavior, proteomics studies are expected to further the current understanding of the m...
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Cancer results from the accumulation of genomic alterations. As the genome is functionally translated to the proteome and regulates tumor cell behavior, proteomics studies are expected to further the current understanding of the molecular mechanisms underlying carcinogenesis and cancer progression. Biomarkers are potential tools to classify cancers for therapy, predict responses to treatments, and support treatment-related decision-making. Biomarker development has been actively pursued in oncology by proteomic approaches. Two-dimensional difference gel electrophoresis (2D-DIGE) is a proteomics technique based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In 2D-DIGE, protein samples are labeled with distinct fluorescent dyes before fractionation via 2D-PAGE. 2D-DIGE offers advantages to identify biomarker candidates, including reproducibility, high sensitivity, comprehensiveness, and high throughput. 2D-DIGE has contributed to the establishment of tissue biomarkers, which potentially facilitate precision medicine. 2D-DIGE is thus expected to yield major advancements in cancer biomarker identification and development.
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A new technique for tube-gel isoelectric focusing (IEF) using plastic tubes instead of glass tubes in the first dimension of two-dimensional gel electrophoresis (2-DE) was developed for proteomic analysis of both denatured and non...
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A new technique for tube-gel isoelectric focusing (IEF) using plastic tubes instead of glass tubes in the first dimension of two-dimensional gel electrophoresis (2-DE) was developed for proteomic analysis of both denatured and non-denatured proteins. The plastic tubes have advantages in handling gels over glass tubes. The plastic tubes were also applied successfully to native gel electrophoresis, blue-native gel electrophoresis, agarose gel IEF, acid-urea gel electrophoresis and cetyltrimethylammonium bromide (CTAB) gel electrophoresis for the first dimension of 2-DE.
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Separation of complex mixtures of proteins by two-dimensional gel electrophoresis (2-DE) is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is cri...
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Separation of complex mixtures of proteins by two-dimensional gel electrophoresis (2-DE) is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for the identification of alterations in protein expression within a given biological system. Despite the availability of several commercially available software packages designed for this purpose, image analysis is extremely resource intensive, subjective and remains a major bottleneck. In addition to reducing throughput, the requirement for manual intervention results in the introduction of operator subjectivity, which can limit the statistical significance of the numerical data generated. A key requirement of image analysis is the accurate definition of protein spot boundaries using a suitable method of image segmentation. We describe a method of spot detection applicable to 2-DE image files using a segmentation method involving pixel value collection via serial analysis of the image through its range of density levels. This algorithm is reproducible, sensitive, accurate and primarily designed to be automatic, removing operator subjectivity. Furthermore, it is believed that this method may offer the potential for improved spot detection over currently available software.
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Most published proteomics studies of bulk plant tissues use a procedure in which proteins are precipitated with trichloroacetic acid (TCA) and acetone (TCA-A), but few attempts have been made to contrast this approach in a systema...
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Most published proteomics studies of bulk plant tissues use a procedure in which proteins are precipitated with trichloroacetic acid (TCA) and acetone (TCA-A), but few attempts have been made to contrast this approach in a systematic way with alternative methods against a spectrum of tissues. To address this, TCA-A was compared with another acetone-based protocol (TCA-B) or a phenol (Phe)-based method, targeting a range of tomato tissues and three species of fruits that contain high levels of contaminating compounds: banana, avocado and orange. The Phe method gave a higher protein yield and typically greater resolution and spot intensity, particularly with extracts from tissues containing high levels of soluble polysaccharides. The methods also generated remarkably different two-dimensional gel electrophoresis (2-DE) protein spot patterns. Peptide mass fingerprinting was used to identify polypeptides that were common to multiple extracts or uniquely present in one extract type. While no clear pattern emerged to explain the basis for the differential protein extraction, it was noted that the Phe method showed enhanced extraction of glycoproteins. These results suggest that the Phe protocol is highly effective with more recalcitrant tissues and that a combination of TCA-A and Phe methods provides enhanced 2-DE based proteomic analyses of most plant tissues.
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The construction of a two-dimensional protein database of the human heart is presented. The database contains information on about 300 abundant proteins of human myocardial tissue, including approximately 40 proteins that were ide...
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The construction of a two-dimensional protein database of the human heart is presented. The database contains information on about 300 abundant proteins of human myocardial tissue, including approximately 40 proteins that were identified by different methods. Each protein was characterized according to several parameters, including molecular weight, isoelectric point, name, partial sequence, subcellular localization, and genetic as well as embryonic changes. [References: 25]
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Proteins from Clostridium pasteurianum were separated by two-dimensional gel electrophoresis. Amino-terminal sequence determination and sequence analysis allowed the identification of 20 proteins, while 11 protein sequences remain...
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Proteins from Clostridium pasteurianum were separated by two-dimensional gel electrophoresis. Amino-terminal sequence determination and sequence analysis allowed the identification of 20 proteins, while 11 protein sequences remained unidentified and one protein appeared to have a blocked amino terminus. [References: 26]
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Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two-dimensional gel electrophoresis, during which immobilized pH 3-10 ...
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Haemophilus influenzae is a bacterium of medical interest of which the entire genome has been sequenced. The proteome of the microorganism has been analyzed by two-dimensional gel electrophoresis, during which immobilized pH 3-10 gradient strips were used and approximately 300 proteins were identified. In order to detect additional, basic proteins, we analyzed the soluble protein fraction of H. influenzae and the proteins of fractions collected from affinity chromatography on heparin, by two-dimensional gel electrophoresis, using for the first-dimensional separation immobilized pH gradient strips comprising the pH region of 6-11. The protein spots were analyzed by matrix-assisted laser desorption ionization-mass spectrometry. One hundred and two proteins were identified, of which 58 were identified for the first time. A large percentage of the basic proteins represent nucleic acid binding and, in particular, ribosomal proteins. The locations of the identified basic proteins of H. influenzae are indicated in a two-dimensional map. [References: 16]
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Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electropho...
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Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobitized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references. [References: 18]
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Velvet mesquite (Prosopis velutina) is a native legume of the southwestern United States and northwestern Mexico, contributing significantly to the desert ecosystem and playing key ecological roles. It is also an important cause o...
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Velvet mesquite (Prosopis velutina) is a native legume of the southwestern United States and northwestern Mexico, contributing significantly to the desert ecosystem and playing key ecological roles. It is also an important cause of allergic respiratory disease widely distributed in the Sonoran, Chihuahuan, and Mojave Deserts. However, no allergens from velvet mesquite pollen have been identified to date. Pollen proteins were extracted and analyzed by one- and two-dimensional electrophoresis and immunoblotting using a pool of 11 sera from mesquite-sensitive patients as the primary antibody. IgE-recognized protein spots were identified by mass spectrometry and bioinformatics analysis. Twenty-four unique proteins, including proteins well known as pollen, food, airway, or contact allergens and four proteins not previously reported as pollen allergens, were identified. This is the first report on allergenic proteins in velvet mesquite pollen. These findings will contribute to the development of specific diagnosis and treatment of mesquite pollen allergy.
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