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A possible approach for gel-based proteomic studies in recalcitrant woody plants

Mónica Sebastiana1, Andreia Figueiredo1*, Filipa Monteiro1, Joana Martins2, Catarina Franco2, Ana Varela Coelho2, Fátima Vaz3, Tânia Simões3, Deborah Penque3, Maria Salomé Pais1 and Sílvia Ferreira1

Author Affiliations

1 Plant Systems Biology Lab, Center of Biodiversity, Functional & Integrative Genomics (BioFIG), Science Faculty of Lisbon University, Lisbon 1749-016, Portugal

2 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da Republica, Oeiras 2780-157, Portugal

3 Laboratório de Proteómica, Departamento de Genética, Instituto Nacional de Saúde Dr. Ricardo Jorge INSA I.P, Lisbon, Portugal

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SpringerPlus 2013, 2:210  doi:10.1186/2193-1801-2-210

Published: 8 May 2013

Abstract

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry.

Keywords:
Grapevine; Pine; Oak; Ectomycorrhizal roots; Protein extraction; 2-DE; Mass spectrometry