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Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.
Front Plant Sci. 2015; 6:620.FP

Abstract

As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes.

Authors+Show Affiliations

Department of Biochemistry and Molecular Biology, University of Cordoba Cordoba, Spain ; Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba Cordoba, Spain ; Centro Multidisciplinario de Investigaciones Tecnológicas, Universidad Nacional de Asunción San Lorenzo, Paraguay.Department of Biochemistry and Molecular Biology, University of Cordoba Cordoba, Spain.Department of Biochemistry and Molecular Biology, University of Cordoba Cordoba, Spain ; Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba Cordoba, Spain.Department of Biochemistry and Molecular Biology, University of Cordoba Cordoba, Spain ; Agricultural and Plant Proteomics Research Group, Department of Biochemistry and Molecular Biology, Escuela Técnica Superior de Ingenieros Agrónomos y de Montes, University of Cordoba Cordoba, Spain.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

26322061

Citation

Romero-Rodríguez, M Cristina, et al. "Multiplex Staining of 2-DE Gels for an Initial Phosphoproteome Analysis of Germinating Seeds and Early Grown Seedlings From a Non-orthodox Specie: Quercus Ilex L. Subsp. Ballota [Desf.] Samp." Frontiers in Plant Science, vol. 6, 2015, p. 620.
Romero-Rodríguez MC, Abril N, Sánchez-Lucas R, et al. Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp. Front Plant Sci. 2015;6:620.
Romero-Rodríguez, M. C., Abril, N., Sánchez-Lucas, R., & Jorrín-Novo, J. V. (2015). Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp. Frontiers in Plant Science, 6, 620. https://doi.org/10.3389/fpls.2015.00620
Romero-Rodríguez MC, et al. Multiplex Staining of 2-DE Gels for an Initial Phosphoproteome Analysis of Germinating Seeds and Early Grown Seedlings From a Non-orthodox Specie: Quercus Ilex L. Subsp. Ballota [Desf.] Samp. Front Plant Sci. 2015;6:620. PubMed PMID: 26322061.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp. AU - Romero-Rodríguez,M Cristina, AU - Abril,Nieves, AU - Sánchez-Lucas,Rosa, AU - Jorrín-Novo,Jesús V, Y1 - 2015/08/11/ PY - 2015/04/07/received PY - 2015/07/27/accepted PY - 2015/9/1/entrez PY - 2015/9/1/pubmed PY - 2015/9/1/medline KW - germination KW - holm oak KW - phosphoproteomics KW - post translational modification KW - recalcitrant seeds SP - 620 EP - 620 JF - Frontiers in plant science JO - Front Plant Sci VL - 6 N2 - As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes. SN - 1664-462X UR - https://www.unboundmedicine.com/medline/citation/26322061/Multiplex_staining_of_2_DE_gels_for_an_initial_phosphoproteome_analysis_of_germinating_seeds_and_early_grown_seedlings_from_a_non_orthodox_specie:_Quercus_ilex_L__subsp__ballota_[Desf_]_Samp_ L2 - https://doi.org/10.3389/fpls.2015.00620 DB - PRIME DP - Unbound Medicine ER -
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