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Wei Wang - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of Three Protein-Extraction Methods for Proteome Analysis of Maize Leaf Midrib, a Compound Tissue Rich in Sclerenchyma Cells.
Frontiers in Plant Science, 2016Co-Authors: Ning Wang, Yanhui Chen, Wei WangAbstract:Leaf morphology is closely related to the growth and development of maize (Zea mays L.) plants and final kernel production. As an important part of the maize leaf, the midrib holds leaf blades in the aerial position for maximum sunlight capture. Leaf midribs of adult plants contain substantial sclerenchyma cells with heavily thickened and lignified secondary walls and have a high amount of Phenolics, making protein Extraction and proteome analysis difficult in leaf midrib tissue. In the present study, three protein-Extraction methods that are commonly used in plant proteomics, i.e., Phenol Extraction, TCA/acetone Extraction, and TCA/acetone/Phenol Extraction, were qualitatively and quantitatively evaluated based on 2DE maps and MS/MS analysis using the midribs of the 10th newly expanded leaves of maize plants. Microscopy revealed the existence of substantial amounts of sclerenchyma underneath maize midrib epidermises (particularly abaxial epidermises). The spot-number order obtained via 2DE mapping was as follows: Phenol Extraction (655) > TCA/acetone Extraction (589) > TCA/acetone/Phenol Extraction (545). MS/MS analysis identified a total of 17 spots that exhibited 2-fold changes in abundance among the three methods (using Phenol Extraction as a control). Sixteen of the proteins identified were hydrophilic, with GRAVY values ranging from -0.026 to -0.487. For all three methods, we were able to obtain high-quality protein samples and good 2DE maps for the maize leaf midrib. However, Phenol Extraction produced a better 2DE map with greater resolution between spots, and TCA/acetone Extraction produced higher protein yields. Thus, this paper includes a discussion regarding the possible reasons for differential protein Extraction among the three methods. This study provides useful information that can be used to select suitable protein Extraction methods for the proteome analysis of recalcitrant plant tissues that are rich in sclerenchyma cells.
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Chloroform-Assisted Phenol Extraction Improving Proteome Profiling of Maize Embryos through Selective Depletion of High-Abundance Storage Proteins
PloS one, 2014Co-Authors: Erhui Xiong, Fangping Gong, Le Yang, Fuju Tai, Wei WangAbstract:The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted Phenol Extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to Phenol Extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and Phenol Extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.
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protein Extraction from plant tissues for 2de and its application in proteomic analysis
Proteomics, 2014Co-Authors: Fangping Gong, Wei WangAbstract:Plant tissues contain large amounts of secondary compounds that significantly interfere with protein Extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein Extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and Phenol Extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and Phenol Extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE-based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).
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universal sample preparation method integrating trichloroacetic acid acetone precipitation with Phenol Extraction for crop proteomic analysis
Nature Protocols, 2014Co-Authors: Erhui Xiong, Wei Wang, Monica Scali, M CrestiAbstract:Crop plants contain large amounts of secondary compounds that interfere with protein Extraction and gel-based proteomic analysis. Thus, a protein Extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein Extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and Phenol Extraction. Through SDS Extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by Phenol Extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and Phenol Extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.
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Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with Phenol Extraction for crop proteomic analysis
Nature protocols, 2014Co-Authors: Erhui Xiong, Wei Wang, Monica Scali, M CrestiAbstract:Crop plants contain large amounts of secondary compounds that interfere with protein Extraction and gel-based proteomic analysis. Thus, a protein Extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein Extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and Phenol Extraction. Through SDS Extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by Phenol Extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and Phenol Extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.
M Cresti - One of the best experts on this subject based on the ideXlab platform.
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universal sample preparation method integrating trichloroacetic acid acetone precipitation with Phenol Extraction for crop proteomic analysis
Nature Protocols, 2014Co-Authors: Erhui Xiong, Wei Wang, Monica Scali, M CrestiAbstract:Crop plants contain large amounts of secondary compounds that interfere with protein Extraction and gel-based proteomic analysis. Thus, a protein Extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein Extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and Phenol Extraction. Through SDS Extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by Phenol Extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and Phenol Extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.
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Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with Phenol Extraction for crop proteomic analysis
Nature protocols, 2014Co-Authors: Erhui Xiong, Wei Wang, Monica Scali, M CrestiAbstract:Crop plants contain large amounts of secondary compounds that interfere with protein Extraction and gel-based proteomic analysis. Thus, a protein Extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein Extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and Phenol Extraction. Through SDS Extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by Phenol Extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and Phenol Extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.
Antonio Morata - One of the best experts on this subject based on the ideXlab platform.
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Electron Beam Irradiation of Wine Grapes: Effect on Microbial Populations, Phenol Extraction and Wine Quality
Food and Bioprocess Technology, 2015Co-Authors: Antonio Morata, Iris Loira, Maria Antonia Banuelos, Wendu Tesfaye, Felipe Palomero, Santiago Benito, María Jesús Callejo, Ana Villa, M. Carmen González, Jose Antonio Suárez-lepeAbstract:Vitis vinifera L. (variety Tempranillo) grapes were subjected to electron beam irradiation at nominal doses of 0.5, 1 and 10 kGy, and the effect on microbial populations, Phenol Extraction and wine quality was examined. No external modifications of fruit shape or colour were observed with any of the doses tested. The 1 kGy dose reduced initial must bacterial and yeast counts by 1 log cycle, while the 10 kGy dose left only a residual population of
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electron beam irradiation of wine grapes effect on microbial populations Phenol Extraction and wine quality
Food and Bioprocess Technology, 2015Co-Authors: Antonio Morata, Iris Loira, Maria Antonia Banuelos, Wendu Tesfaye, Felipe Palomero, Santiago Benito, María Jesús Callejo, Ana Villa, Carmen M Gonzalez, Jose Antonio SuarezlepeAbstract:Vitis vinifera L. (variety Tempranillo) grapes were subjected to electron beam irradiation at nominal doses of 0.5, 1 and 10 kGy, and the effect on microbial populations, Phenol Extraction and wine quality was examined. No external modifications of fruit shape or colour were observed with any of the doses tested. The 1 kGy dose reduced initial must bacterial and yeast counts by 1 log cycle, while the 10 kGy dose left only a residual population of <10 colony-forming units (cfu)/mL. Irradiation was associated with a dose-dependent increase in Phenolic compounds in the must. However, the wines produced from grapes irradiated at different doses showed no significant differences in their total Phenolic compound contents. All the wines had a good sensory profile; those irradiated at 10 kGy had an increased fruity odour.
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grape processing by high hydrostatic pressure effect on microbial populations Phenol Extraction and wine quality
Food and Bioprocess Technology, 2015Co-Authors: Antonio Morata, Iris Loira, Ricardo Vejarano, Maria Antonia Banuelos, P D Sanz, Laura Otero, Jose Antonio SuarezlepeAbstract:Vitis vinifera (variety Tempranillo) grapes were subjected to high hydrostatic pressure (HHP) treatments of 200, 400 and 550 MPa for 10 min, and its effect on microbial populations, Phenol Extraction and wine quality was examined. At ≥400 MPa, the wild yeast population was strongly reduced from 104 to <10 cfu/ml. Bacteria showed greater resistance, and a residual load remained even after the treatment at 550 MPa. The Extraction of Phenolic compounds from the HHP-treated grapes was improved, with higher concentrations of total Phenols obtained compared to crushing alone. Anthocyanin Extraction was also increased, producing wines with better colour intensity. These wines also had higher methanol and ethanol contents and returned higher aromatic quality and colour scores. The HHP treatment of grapes may assist in the use of yeast starters, increase Phenol Extraction from grape skins and improve wine quality.
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Grape Processing by High Hydrostatic Pressure: Effect on Microbial Populations, Phenol Extraction and Wine Quality
Food and Bioprocess Technology, 2015Co-Authors: Antonio Morata, Iris Loira, Ricardo Vejarano, Maria Antonia Banuelos, P D Sanz, Laura Otero, Jose Antonio Suárez-lepeAbstract:Vitis vinifera (variety Tempranillo) grapes were subjected to high hydrostatic pressure (HHP) treatments of 200, 400 and 550 MPa for 10 min, and its effect on microbial populations, Phenol Extraction and wine quality was examined. At ≥400 MPa, the wild yeast population was strongly reduced from 10^4 to
Erhui Xiong - One of the best experts on this subject based on the ideXlab platform.
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Chloroform-Assisted Phenol Extraction Improving Proteome Profiling of Maize Embryos through Selective Depletion of High-Abundance Storage Proteins
PloS one, 2014Co-Authors: Erhui Xiong, Fangping Gong, Le Yang, Fuju Tai, Wei WangAbstract:The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted Phenol Extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to Phenol Extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and Phenol Extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.
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universal sample preparation method integrating trichloroacetic acid acetone precipitation with Phenol Extraction for crop proteomic analysis
Nature Protocols, 2014Co-Authors: Erhui Xiong, Wei Wang, Monica Scali, M CrestiAbstract:Crop plants contain large amounts of secondary compounds that interfere with protein Extraction and gel-based proteomic analysis. Thus, a protein Extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein Extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and Phenol Extraction. Through SDS Extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by Phenol Extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and Phenol Extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.
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Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with Phenol Extraction for crop proteomic analysis
Nature protocols, 2014Co-Authors: Erhui Xiong, Wei Wang, Monica Scali, M CrestiAbstract:Crop plants contain large amounts of secondary compounds that interfere with protein Extraction and gel-based proteomic analysis. Thus, a protein Extraction protocol that can be easily applied to various crop materials with minimal optimization is essential. Here we describe a universal protocol for total protein Extraction involving trichloroacetic acid (TCA)/acetone precipitation followed by SDS and Phenol Extraction. Through SDS Extraction, the proteins precipitated by the TCA/acetone treatment can be fully resolubilized and then further purified by Phenol Extraction. This protocol combines TCA/acetone precipitation, which aggressively removes nonprotein compounds, and Phenol Extraction, which selectively dissolves proteins, resulting in effective purification of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues, and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be completed within 5 h.
Jose Antonio Suarezlepe - One of the best experts on this subject based on the ideXlab platform.
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electron beam irradiation of wine grapes effect on microbial populations Phenol Extraction and wine quality
Food and Bioprocess Technology, 2015Co-Authors: Antonio Morata, Iris Loira, Maria Antonia Banuelos, Wendu Tesfaye, Felipe Palomero, Santiago Benito, María Jesús Callejo, Ana Villa, Carmen M Gonzalez, Jose Antonio SuarezlepeAbstract:Vitis vinifera L. (variety Tempranillo) grapes were subjected to electron beam irradiation at nominal doses of 0.5, 1 and 10 kGy, and the effect on microbial populations, Phenol Extraction and wine quality was examined. No external modifications of fruit shape or colour were observed with any of the doses tested. The 1 kGy dose reduced initial must bacterial and yeast counts by 1 log cycle, while the 10 kGy dose left only a residual population of <10 colony-forming units (cfu)/mL. Irradiation was associated with a dose-dependent increase in Phenolic compounds in the must. However, the wines produced from grapes irradiated at different doses showed no significant differences in their total Phenolic compound contents. All the wines had a good sensory profile; those irradiated at 10 kGy had an increased fruity odour.
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grape processing by high hydrostatic pressure effect on microbial populations Phenol Extraction and wine quality
Food and Bioprocess Technology, 2015Co-Authors: Antonio Morata, Iris Loira, Ricardo Vejarano, Maria Antonia Banuelos, P D Sanz, Laura Otero, Jose Antonio SuarezlepeAbstract:Vitis vinifera (variety Tempranillo) grapes were subjected to high hydrostatic pressure (HHP) treatments of 200, 400 and 550 MPa for 10 min, and its effect on microbial populations, Phenol Extraction and wine quality was examined. At ≥400 MPa, the wild yeast population was strongly reduced from 104 to <10 cfu/ml. Bacteria showed greater resistance, and a residual load remained even after the treatment at 550 MPa. The Extraction of Phenolic compounds from the HHP-treated grapes was improved, with higher concentrations of total Phenols obtained compared to crushing alone. Anthocyanin Extraction was also increased, producing wines with better colour intensity. These wines also had higher methanol and ethanol contents and returned higher aromatic quality and colour scores. The HHP treatment of grapes may assist in the use of yeast starters, increase Phenol Extraction from grape skins and improve wine quality.