Reduction of proteins during sample preparation and two‐dimensional gel electrophoresis of woody plant samples

Vâlcu, Cristina-Maria; Schlink, Katja

doi:10.1002/pmic.200500314

Cited by 36 publications

(35 citation statements)

References 55 publications

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“…It reacts to reduce cystines stoichiometrically at low millimolar concentrations [36]. Vâlcu and Schlink [33] optimized the reducing agent content in lysis buffers on woody plant tissues including beech leaves and roots and spruce needles, and found optimal extraction was achieved with 65 mM DTT for leaves and needles and a mixture of 2 mM TCEP and 50 mM DTT for roots.…”

Section: Protein Solubilizationmentioning

confidence: 99%

See 1 more Smart Citation

Optimizing protein extraction from plant tissues for enhanced proteomics analysis

Wang¹,

Tai

Chen

2008

J of Separation Science

148

110

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Plant tissues usually contain high levels of proteases and secondary metabolites that severely interfere with protein extraction, separation, and identification. Preparation of high-quality protein samples from plant tissues for proteomic analysis represents a great challenge. This article briefly describes the critical points in protein separation, especially secondary metabolites in plant tissues, and removal strategy. It provides an updated overview of three total protein extraction methods and their applications in proteomic analysis of various recalcitrant tissues.

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Section: Protein Solubilizationmentioning

confidence: 99%

“…For example, phenolics can build irreversible complexes with proteins, and the oxidation of phenolics by phenoloxidases and peroxidases can cause streaking and generate artefactual spots on 2-DE gels [33]. Pigments, polysaccharides, and lipids can also cause severe disturbances in 2-DE gels [31].…”

Section: Secondary Metabolites and Their Removalmentioning

confidence: 99%

Optimizing protein extraction from plant tissues for enhanced proteomics analysis

Wang¹,

Tai

Chen

2008

J of Separation Science

148

110

View full text Add to dashboard Cite

show abstract

“…These compounds can produce protein precipitation when the tissues are disturbed, and they can interfere severely with 2D-E separation when they co-precipitate with proteins [23,24]. In order to achieve the best representation of wild cardoon flowers proteome, the use of an effective protein extraction method that delivered fewer interfering substances, the greatest number of distinct proteins and more clearly protein spots in 2D gel, was essential.…”

Section: Resultsmentioning

confidence: 99%

“…Furthermore, three spots were found at low molecular levels, with an approximate molecular mass in the range of 10 and 20 kDa. In fact, according to several studies of cardoon's protein profile [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], proteins with low Mw, represented the β-subunits of cardosins (16.5 and 13.5 kDa), since there were a positive correlation between band intensities and milk-clotting activities.…”

Section: Protein Separation On 2d-e and Maldi-tof-ms Identificationmentioning

confidence: 99%

Identification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach

et al. 2016

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Proteomic approach was applied to identify total proteins, particularly the enzymatic content, from wild cardoon flowers. As the selection of an appropriate sample preparation method is the key for getting reliable results, two different extraction/precipitation methods (trichloroacetic acid and phenol/ammonium acetate) were tested on fresh and lyophilized flowers. After two-dimensional electrophoresis (2D-E) separations, a better protein pattern was obtained after phenol extraction from lyophilized flowers. Only 46 % of the total analyzed spots resulted in a protein identification by mass spectrometry MALDI-TOF. Four proteases (cardosins A, E, G, and H), which have become a subject of great interest in dairy technology, were identified. They presented molecular weights and isoelectric points very close and high levels of homology between matched peptides sequences. The absence of the other cardosins (B, C, D, and F) could be an advantage, as it reduces the excessive proteolytic activity that causes bitter flavors and texture defects, during cheese making.

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“…These substances interfere with the resolution of the resulting protein profiles on SDS-PAGE gel or 2-DE. For example, phenolic compounds can bind to proteins causing latitude lines and patches of shadows on the 2-DE gels (Vâlcu and Schlink, 2006).…”

Section: Protein Extraction Without Tca/acetone Precipitation Producementioning

confidence: 99%

A simple protein extraction method for proteomic analysis of mahogany (Swietenia macrophylla) embryos

et al. 2017

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In any proteomic studies, protein extraction and sample preparation are the most crucial steps for obtaining optimal results. This is to ensure extracted proteins are not only high in yield but also clean from contaminants that could affect downstream proteomic applications such as two dimensional gel electrophoresis (2-DE) and mass spectrometry. Tissues from plants and trees such as Swietenia macrophylla are often rich in non-protein contaminating substances, which could interfere in the proteomic applications. S. macrophylla or also known as the mahogany is one of the most valuable tree species in the world. Studies on proteins for this tree as well as its seeds are very limited. We have extracted proteins from S. macrophylla seeds (specifically embryo tissues) using three different methods, each having different lysis buffer recipes. Furthermore, another set of samples were precipitated using trichloroacetic acid/acetone prior to the three extraction methods to further purify the protein samples. The results from 2-DE analysis showed approximately 240 protein spots were detected from the successful protocol using a lysis buffer of 9 M urea, 4% CHAPS, 0.5% triton X-100 and 100 mM DTT without TCA/acetone precipitation. This study highlights the aspects of sample preparation for S. macrophylla embryos, focusing on the total protein extraction and resolution in SDS-PAGE as well as 2-DE. Furthermore, this is the very first report of the proteome 2DE profile from S. macrophylla embryo.

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Reduction of proteins during sample preparation and two‐dimensional gel electrophoresis of woody plant samples

Cited by 36 publications

References 55 publications

Optimizing protein extraction from plant tissues for enhanced proteomics analysis

Optimizing protein extraction from plant tissues for enhanced proteomics analysis

Identification of proteins from wild cardoon flowers (Cynara cardunculus L.) by a proteomic approach

A simple protein extraction method for proteomic analysis of mahogany (Swietenia macrophylla) embryos

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