title: A possible approach for gel-based proteomic studies in recalcitrant woody plants.
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.
authors.affiliation: Plant Systems Biology Lab, Center of Biodiversity, Functional & Integrative Genomics (BioFIG), Science Faculty of Lisbon University, Lisbon, 1749-016 Portugal.
authors.forename: Mónica
authors.initials: M
authors.lastname: Sebastiana
authors.forename: Andreia
authors.initials: A
authors.lastname: Figueiredo
authors.forename: Filipa
authors.initials: F
authors.lastname: Monteiro
authors.forename: Joana
authors.initials: J
authors.lastname: Martins
authors.forename: Catarina
authors.initials: C
authors.lastname: Franco
authors.forename: Ana Varela
authors.initials: AV
authors.lastname: Coelho
authors.forename: Fátima
authors.initials: F
authors.lastname: Vaz
authors.forename: Tânia
authors.initials: T
authors.lastname: Simões
authors.forename: Deborah
authors.initials: D
authors.lastname: Penque
authors.forename: Maria Salomé
authors.initials: MS
authors.lastname: Pais
authors.forename: Sílvia
authors.initials: S
authors.lastname: Ferreira
keywords: 2-DE; Ectomycorrhizal roots; Grapevine; Mass spectrometry; Oak; Pine; Protein extraction