Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer–based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta. The detergent-free phenol-based extraction combined with dodecyl-β-d-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-d-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.
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R. serbica transcriptome database, publically available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta
R. serbica protein fasta file: http://www.biomed.unipd.it/filearrigoni/Ramonda_Serbica_Protein_fasta_file.fasta
Filter-aided sample preparation
Fully hydrated leaves
Polyacrylamide gel electrophoresis
Photosynthetic electron transport chain
Relative water content
Sodium dodecyl sulphate
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The authors would like to acknowledge the technical help provided Dr. Anna Rita Trentin (DAFNAE, University of Padova) and Ilaria Battisti (Proteomics Center University of Padova and Azienda Ospedaliera di Padova). The authors wish to thank the Cassa di Risparmio di Padova e Rovigo (Cariparo) Holding for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer.
This work was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia (contract no. 451-03-68/2020-14/200053), the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663), and the University of Padova (grant number BIRD189887/18 to G.A). M.Vidović received financial support from COST Action BM1405 (STSM-BM1405-190218-092344 and STSM-BM1405-190317-080965).
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Vidović, M., Franchin, C., Morina, F. et al. Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants. Anal Bioanal Chem 412, 8299–8312 (2020). https://doi.org/10.1007/s00216-020-02965-2
- Phenol-based extraction
- Peptide LC-MS/MS analysis
- Ramonda serbica
- Recalcitrant plant material
- Resurrection plants
- Soluble and membrane-bound protein extraction