Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica
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RESEARCH PAPER
Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants Marija Vidović 1,2 & Cinzia Franchin 3,4 & Filis Morina 1,5 & Sonja Veljović-Jovanović 1 & Antonio Masi 6 & Giorgio Arrigoni 3,4,7 Received: 4 April 2020 / Revised: 31 August 2020 / Accepted: 22 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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. Keywords Phenol-based extraction . Peptide LC-MS/MS analysis . Ramonda serbica . Recalcitrant plant material . Resurrection plants . Soluble and membrane-bound protein extraction
Abbreviations βME 2-Mercaptoethanol CHAPS
DDM
3-[(3-Cholamidopropyl)dimethylammonio]-1propanesulphonate Dodecyl-β-D-maltoside
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02965-2) contains supplementary material, which is available to authorized users. * Marija Vidović [email protected] * Giorgio Arrigoni [email protected]
4
Proteomics Center University of Padova and Azienda Ospedaliera di Padova, Via G. Orus 2/B, 35129 Padua, Italy
5
Present address: Department of Plant Biophysics & Biochemistry, Biology Centre, Institute of Plant Molecular Biology, Czech Academy of Sciences, Branišovská 31/1160, 37005 České Budějovice, Czech Republic
1
Institute for Multidisciplinary Research, Department of Life Science, Uni
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